A sequencing road trip at the heartland of Zika virus in the Americas

A year ago, on the 2nd June 2016, our adventure began in northeast Brazil.

Go to the profile of Nuno Rodrigues Faria
May 25, 2017
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We had this ambitious plan to travel 2,000 km by road from Natal to Salvador investigating the Zika virus epidemic in the country. This journey had a name - ZiBRA - an acronym for Zika in Brazil Real-time Analysis. We would travel with all the equipment and skills required to perform diagnostics, sequencing, mosquito identification in real-time. The multidisciplinary team from FIOCRUZ Bahia, Instituto Evandro Chagas and the universities of São Paulo, Birmingham and Oxford had specialists in molecular biology, bioinformatics, entomology and genomic epidemiology.

First group meeting in Natal, Rio Grande do Norte, 2nd June 2016. Early morning we would start with a group meeting to address what were the main goals of the day.

The project kicked-off with a 2-day testing-and-sequencing marathon in the public health laboratory (LACEN) headed by Dr. Maria de Queiroz in Natal, capital city of the Rio Grande do Norte state, one of the 9 states in the northeast region of Brazil. After the first reports associating Zika virus and microcephaly in this region in October 2015, many pregnant women presenting with clinical symptoms compatible with Zika virus infection visited their local health facility, and these samples eventually arrived to Dr. Queiroz’s lab for testing. The northeast region was the most affected by Zika virus and microcephaly, and to help with the huge demand for molecular diagnostic testing we analysed >1300 samples in laboratories across the capital cities of Paraíba, Pernambuco, Alagoas and Bahia states. While on the road, quantitative PCR results - averaging 150 tests per day - were sent to the directors of each LACEN and to the Brazilian Ministry of Health (MoH).

Ingra Morales Claro (University of Sao Paulo, Brazil) and Marta Giovanetti (FIOCRUZ Bahia, Brazil) preparing sequencing libraries inside the minibus.

With little more than a handful of Zika virus genomes available back then, we wanted to provide a better understanding of the origins and to the elucidate the main drivers of the Zika epidemic in the Americas (Faria et al Science 2016). For this we needed to sequence more Zika genomes from archived and present day samples and collect all the available epidemiological data. The epidemiological data was obtained with the MoH. The genomic data was generated in country using an updated version of the method used to sequence Ebola virus genomes in real-time during the 2014 West African outbreak (Quick et al., Nature).

Antonio Charlys da Costa (University of Sao Paulo, Brazil) stands by the minibus and supporting ZiBRA van (credits to Nuno Faria). 

The miniaturisation of sequencing technology in recent years by Oxford Nanopore Technologies have made real-time monitoring of ongoing outbreaks possible. This way a researcher with little more than a suitcase can achieve what previously required a whole specialised laboratory to achieve. The biggest challenge for the laboratory team was teasing out the tiny amount of viral genetic material contained within the clinical samples, typically blood. Zika virus causes a relatively mild infection compared to some of its arbovirus relatives. This means small numbers of viral particles circulating in the bloodstream which in turn makes detection more challenging. In order to recover as much of the genetic information as possible we used an amplification method called multiplex PCR. PCR is highly sensitive yet prefers small targets, so to recover the whole genome we had to develop a method to perform many small overlapping reactions in a single tube simultaneously. The current best method at any time was available on the ZiBRA project website for others to use (http://zibraproject.org) and suggest improvements to. The final version of the protocol is available here (Quick et al. Nature Protocols).

Poliana Lemos, Bruna Nascimento and Hamilton Monteiro from (Evandro Chagas Institute, Brazil) capturing mosquitoes during the ZiBRA trip.

Using the newly generated data our team estimated that the critical turning point in the emergence of Zika virus in the Americas was the introduction of Zika to northeast Brazil in or around February 2014, more than a year from its first detection in May 2015. From there the virus travelled to other regions in South and Central America, and also to the Caribbean. These migration events most likely occurred 1 year before the earliest date of confirmation in each recipient location.

That Zika was circulating silently across most of the Americas highlights the need for a better surveillance system for Zika virus across the globe. There is also a need to extend genomic surveillance to other pathogens. For example, following the invitation from the MoH, between the 24 Apr - 1 May 2017 we joined and trained local teams in the LACEN of Minas Gerais and we have generated 20 novel genomes from the recently re-emerged yellow fever virus in Brazil (virological.org).

We are currently writing from Manaus, capital city of the Amazonas state on the north region of Brazil. New team members from Manaus, Rio de Janeiro and Minas Gerais are being trained in nanopore genome sequencing as we write. Within 36 hours of arrival we generated 3 libraries to sequence 16 Zika virus genomes demonstrating how rapidly a mobile lab can be deployed. Several of the young and talented Brazilian scientists will also be joining our labs soon to extend the methods further and work on other arbovirus research projects in Brazil and elsewhere. The final goal? To develop a sustainable real-time genomic and epidemiological surveillance network across institutions and geographic locations, capable of informing effective public health strategies against Zika and other viruses in the Americas.

Our Nature paper is here: http://go.nature.com/2qGwtjA
Our Nature Protocols paper is here: http://go.nature.com/2rw1msb

A related study by Metsky et al. in Nature, is also published today: 


As well as a News & Views article on the four related Zika papers: http://go.nature.com/2rk6Qqb

Nuno Faria and Josh Quick contributed equally to this post


Go to the profile of Nuno Rodrigues Faria

Nuno Rodrigues Faria

Sir Henry Dale Fellow at University of Oxford, University of Oxford, Department of Zoology


Go to the profile of Nonia Pariente
Nonia Pariente about 2 months ago

Great post Nuno! I'm always amazed by the speed and portability of the existing technology and your approach of empowering the local communities to do the analyses autonomously will make this a stable procedure for outbreaks to come. Many congratulations to all involved in all papers!