Innovative handheld genetic sequencing device helps scientists track evolution of Zika virus

An international group of researchers on board a mobile laboratory equipped with an innovative handheld genetic sequencing device are tracking the movement of the Zika virus since it disembarked in Brazil and began spreading through the Americas.

According to the scientists, their aim is to monitor the viral genome's evolution and to understand what has happened, so as to be able to predict future outbreaks and to keep diagnostic methods up to date.

The first results of Project ZiBRA (Zika in Brazil Real Time Analysis), which is supported by Brazil's Ministry of Health, FAPESP and several other entities, were published in the journal Nature.

"By combining epidemiological and genetic data, we were able to see that Zika circulated silently in all regions of the Americas at least a year before the virus was first confirmed, in May 2015," said Nuno Faria, a researcher in the Zoology Department of Oxford University, UK, and first author of the article.

According to Faria, Zika was introduced into Brazil's Northeast region in February 2014. Transmission in the region probably occurred throughout the year but was not especially pronounced.

"The major outbreak very probably occurred in 2015, concurrently with the dengue outbreak," he said. "Zika spread from Northeast to Southeast Brazil [initially Rio de Janeiro] and also to the Caribbean and other countries in South and Central America, eventually reaching Florida."

The findings were based on an analysis of 254 whole genomes of the pathogen, 54 of them sequenced in this study. Most of the new genetic data were obtained using MinION, a palm-sized sequencer made by Oxford Nanopore Technologies and weighing less than 100 g.

The protocols that enabled this technology to sequence Zika were developed as part of Project ZiBRA and gave rise to a second article that was also published in the journal Nature Protocols.

"The test was first used in 2015 in Africa, during the Ebola epidemic. Its main advantage is that it can be performed at the site where a case has been notified, so that the virus's trajectory can be tracked in real time. The device is smaller than a smartphone and can sequence the whole genomes of microorganisms. Sequencing of larger organisms will be possible soon," said Ester Sabino, a researcher at the University of São Paulo's Tropical Medicine Institute (IMT-USP) in São Paulo, Brazil, and a co-author of the article.

The larger the number of sequences generated, the easier it becomes to understand when the virus entered Brazil, how it spread across the continent, and, above all, how it is evolving, Sabino added.

The analysis is made possible by the molecular clock technique, which evaluates substitutions in certain gene sequences. These changes occur at a relatively constant rate, with the genes comparable to clocks in that their mutations indicate when different viral isolates diverged.

"The idea for the project came up in 2016, when the first epidemiological and genetic findings on Zika in the Americas were published by part of the group in the journal Science. At the time, we had sequenced seven viral isolates. The number of samples was insufficient to give us a broad notion of Zika's diversity on the continent," said Luiz Carlos Alcântara from Oswaldo Cruz Foundation (FIOCRUZ) in Bahia State, northeastern Brazil.

ZiBRA was approved in a joint call for proposals issued by three British research funding agencies: the UK Medical Research Council, the Newton Fund, and the Wellcome Trust. The original proponents were joined by researchers funded by several Brazilian institutions - the National Council for Scientific & Technological Development (CNPq), FAPESP, FIOCRUZ, Evandro Chagas Institute, the Ministry of Health and USP - and others from Birmingham University and Oxford University, UK.

A mobile lab was installed in a bus and, in 2016, visited Central Public Health Laboratories in four northeastern states: Rio Grande do Norte, Paraíba, Pernambuco and Alagoas. Besides Alcântara, Faria and Sabino, the initiative was coordinated by researchers Nick Loman, from Birmingham University's School of Biosciences; Oliver Pybus, from Oxford University's Zoology Department; and Marcio Nunes, from Evandro Chagas Institute in Pará, Brazil.

"At each Central Lab, we analyzed 300-400 blood samples from patients with suspected Zika virus infections, performing a total of 1,330 tests. The diagnostic technique was real-time PCR [which detects viral RNA in the sample]. When the result was positive, the virus's genetic material was sequenced," Alcântara said.

With support from two fixed labs, at FIOCRUZ in Salvador, Bahia, and IMT-USP in São Paulo, isolates from the Southeast region and from Tocantins State were also sequenced.

The genomes of four viral isolates from Mexico and five from Colombia were sequenced in the US by scientists who collaborated with the group, also as part of Project ZiBRA.

"The analyses showed that the viruses found in the various regions of Brazil and other Latin American countries had not yet undergone many mutations or developed significant diversity," Alcântara said. "However, in light of what's been observed in Asia, the virus is likely to be very different in the near future, so we have to continue monitoring developments. If the tests used for diagnosis don't keep up with the virus's evolution, they may become ineffective."

According to Alcântara, the virus appears to have arrived in Asia from Africa shortly before 2007, when it caused the first major epidemic in Micronesia. Fresh outbreaks occurred in the Philippines (2012) and French Polynesia (2013 and 2014). It is then believed to have arrived in Brazil, which has reported the largest number of cases to date: more than 200,000 by December 2016, according to the article.

"The virus has changed a good deal since it left Africa," Alcântara said. "Its diversity here in the Americas will probably be much greater in seven or ten years' time. We must maintain genomic surveillance in order to be prepared for a new outbreak if and when it comes."

In addition to helping Central Labs diagnose hundreds of suspected Zika cases in 2016, the ZiBRA researchers also trained the teams that took part in tests during the road trip to perform genomic surveillance using the MinION portable sequencer.

Alcântara is now heading the second stage of the project, which involves monitoring dengue, chikungunya and yellow fever, as well as Zika.



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