When health officials confirmed the first locally acquired Zika cases in the continental United States in Miami in July 2016, they knew in a general way how the mosquito-borne virus probably arrived.
The Aedes aegypti mosquitoes that serve as the vector between Zika and humans are present in Miami, but their range is limited — they don’t travel farther than about 1½ football fields. Tourists do.
Since none of the Florida cases involved people who had visited an affected country or had sex with someone who did, a traveler must have introduced the virus to local mosquitoes after being bitten abroad and then again in Miami.
Now a new study by a large group of international researchers sheds more light on how and when that happened. The study found the virus was introduced into Miami in 2016 at least four and up to 40 separate times, with most of the viral lineages from strains found in the Caribbean.
Co-led by scientists at Fred Hutchinson Cancer Research Center, researchers sequenced the genomes from infected patients and mosquitoes from different times in the Miami outbreak — which reached about 250 cases — and created a phylogenetic tree, or genetic history, of the viruses.
“By working with the genome sequences, we were able to figure out how closely related these cases were and how many introduction events there were,” said Fred Hutch evolutionary biologist Dr. Trevor Bedford, one of more than 60 scientists who contributed to the study. The study was co-led by researchers from The Scripps Research Institute, the U.S. Army Medical Research Institute of Infectious Diseases, Florida Gulf Coast University, the University of Oxford, the Florida Department of Health and the Broad Institute of MIT and Harvard.
The Miami research was one of three related studies published Wednesday in the journal Nature that analyzed Zika transmission and evolution using genomic sequencing. In the two others:
A fourth study published in Nature Protocols detailed the technologies used by all the research teams.
Rapid genome sequencing during ongoing outbreaks has only been possible in recent years because of new sequencing technologies, according to Scripps’ Dr. Kristian G. Andersen, a senior author of the Miami paper, who contributed to all three papers and also worked on the 2013–2016 Ebola epidemic in West Africa. Combined with an ethos for “open science,” or sharing data in real time, these new tools are telling researchers more about outbreaks than they can learn through standard surveillance.
They also relay valuable lessons on how to respond to or even prevent the next Zika outbreak.
That as few as four travelers led to the infection of more than 250 people in Miami, for example, meant that there was considerable spread by local mosquitoes, said Bedford, a finding that underscored the need for mosquito control. Miami used aerial spraying and sent workers house to house to destroy breeding sites, which the study found correlated with a drop in cases.
“Finding that there was considerable local spread, vector control is the way to control this particular outbreak,” Bedford said.
The evolutionary trees also suggested that Zika transmission in Florida may have started at least two months before it was first detected in July, the study found — a valuable heads-up to health officials hoping to halt an outbreak this year.
After researchers noted that three of the four lineages sequenced in Miami clustered with genomes sequenced from the Dominican Republic and Guadeloupe, Dr. Gytis Dudas, a Mahan Postdoctoral Fellow at Fred Hutch and contributor to the Miami study, sought to corroborate this finding by investigating other data. He started with travel patterns.
Miami received more international air and sea traffic than any other city in the continental U.S. in 2016, and between January and June, more than half of those travelers — 3 million— came from the Caribbean. With its year-round population of Aedes aegypti, Miami was a “perfect storm” for Zika transmission, according to Dr. Nathan D. Grubaugh, a Scripps research associate and the Miami study’s lead author.
While the Miami paper provided a dense sampling of a small, localized outbreak, the Broad group sequenced complete or partial genomes from 100 samples in 10 countries and combined them with 64 already available genomes to paint a big-picture view of Zika’s rapid spread through the Americas to the Caribbean.
The Broad team’s analysis of Zika’s spread to the Caribbean also corroborated the Miami study’s findings.
“With the Caribbean samples sequenced by the Broad, we were able to connect the dots in Florida,” said Bedford.
As in the Miami study, the genetic histories constructed by the Broad researchers revealed the limits of traditional surveillance systems to detect Zika.
"One of the important findings of the paper is that Zika virus circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed,” said Fred Hutch biostatistician Dr. Elizabeth “Betz” Halloran, who contributed to the Broad paper.
Determining when Zika virus arrived in specific regions, the Broad authors wrote, tells health officials when and where to look for rising incidence of possible complications of Zika infection.
For up to 80 percent of those infected, Zika causes mild or no symptoms. But for pregnant women, infection can have devastating consequences. In Brazil, thousands of babies have been born with microcephaly — a tiny head with a severely underdeveloped brain — and other developmental deficits. In adults, Zika also is rarely associated with Guillain-Barré syndrome, which may result in paralysis.
Real-time sequencing came into its own during the West African Ebola epidemic. Relatively early in the outbreak, researchers sequenced Ebola genomes from patient blood samples and immediately uploaded them to the public database GenBank, leading to a surge of collaboration from experts in diverse fields. The collection of shared, publicly available data helped confirm probable transmission routes and other information crucial to public health interventions.
But as was noted in the Broad study, sequencing the Zika genome has been more challenging than sequencing Ebola. Zika virus is present in lower levels than Ebola in clinical samples and its genetic material degrades rapidly. Despite hundreds of thousands of cases across the Americas, researchers started out with few genomes to analyze.
To remedy that, scientists from around the world went to Brazil in June 2016 for a sequencing road trip, traveling through northeast Brazil with a mobile genome lab. Bedford took part in the project, which was dubbed ZiBRA, for Zika in Brazil Real-Time Analysis. It was led by Prof. Ester Sabino of the University of São Paulo, Prof. Luiz Alcantara of the Oswaldo Cruz Foundation (Fiocruz) Salvador, Prof. Nick Loman of Britain’s University of Birmingham, and Dr. Nuno Faria and Prof. Oliver Pybus of Oxford University. Prior to departure, Loman and Josh Quick, a University of Birmingham doctoral student, developed an initial protocol to amplify low levels of virus for sequencing from clinical material.
With scientists from Fiocruz Salvador, the University of São Paulo and the Instituto Evandro Chagas and with support provided by the Brazilian Ministry of Health, the group traveled by bus to various government diagnostic laboratories, putting in 13-hour days in the lab.
“This was an amazing team to get to be a part of,” Bedford said.
Ingra Morales, a doctoral student from Sabino’s laboratory at the University of São Paulo, came on the trip to help sequence Zika genomes. But she and the others quickly realized that the immediate task was helping laboratories perform molecular diagnostics on a backlog of clinical samples to try to understand the full extent of Zika infections over the past year.
“As we passed through the cities, we saw that the study was of paramount importance because in addition to genetic data, we could also assist [the laboratories] in molecular diagnostics,” she said in an email from Brazil. “In less than 48 hours we had the results of many patient samples, which we provided to the local public health laboratories.”
Sequencing was carried out in real time on a mobile laboratory bus. Initial results were promising, but whole genome sequences were hard to achieve.
“The tiny amounts of Zika virus found in samples was surprising and necessitated developing a new sequencing approach to give the exquisite sensitivity required,” said Quick, who spent the summer months after the trip refining the approach.
Allison Black, a University of Washington doctoral student in epidemiology who works in Bedford’s lab, headed from Seattle to northeast Brazil in September to help with the sequencing, joining Morales, three other Brazilian researchers and Quick in humid Salvador.
As before, the group would head out early every morning and put in long, coffee-fueled days in the lab, applying the newly refined approach to amplify the small amount of virus they had to work with in each sample.
“This was an entirely new technique, so we didn’t know if it would work well until we tested it on real samples, but thankfully it worked splendidly,” Black said.
The results were impressive: The team was able to sequence 54 genomes when only 90 from the hard-to-sequence virus had been publicly available before. One of their findings was a sequence representing the earliest confirmed Zika infection in Brazil.
The genetic history allowed researchers to more accurately assess when Zika was introduced to Brazil and how it spread after its introduction, important steps toward understanding the scope of the Brazilian epidemic. Such information is hard to get from surveillance alone because Zika symptoms, if present at all, can be difficult to distinguish from other viruses circulating, said Black.
According to Morales, the project underscored the importance of not only working together during an outbreak but also sharing that work openly and widely.
“We are living in a moment where new technologies allow the rapid sharing of information that can contribute to other studies and help other people or outbreaks,” she said via email. “During the ZiBRA project, all the information and protocols gathered were shared in real time at www.zibraproject.org or on social networks like Twitter and Facebook, and we were able to see people commenting, giving suggestions, opinions and using what worked for us. This is amazing.”
The research is continuing, led by Alcantara, who aims to define the genetic diversity of Zika and other mosquito-borne infections across all Brazil, starting with the ZiBRA2 project in the Amazon rainforest region this week.
Mary Engel is a former staff writer at Fred Hutchinson Cancer Research Center. Previously, she covered medicine and health policy for the Los Angeles Times, where she was part of a team that won a Pulitzer Prize for Public Service. She was also a fellow at the Knight Science Journalism Program at MIT. Follow her on Twitter @Engel140.