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Genomic Studies Untangle Zika Virus Introduction, Spread Across Americas

NEW YORK (GenomeWeb) – Three independent research teams took a genomic look at the Zika virus (ZIKV) for a collection of interrelated studies, using sequences from hundreds of clinical and Aedes aegypti mosquito ZIKV samples to track the origins and spread of the 2015 Zika outbreak.

"As the outbreak erupted and spread, it became obvious that, amongst the many gaps in our knowledge about the virus, there was a dearth of information about Zika [virus] genetic diversity and big open questions that this could help address, like how the virus spreads through populations and changes as it goes," Bronwyn MacInnis, associate director of malaria and viral genomics at the Broad Institute's Infectious Disease and Microbiome Program, said during a telephone press briefing this week.

For the one of the three studies published in Nature today, MacInnis and her colleagues from the Broad Institute led a team that sequenced 110 ZIKV isolates collected across 10 countries, pinpointing the events associated not only with the rapid bursts in ZIKV infections in Brazil, but also epidemic strain introductions to places such as Puerto Rico, Honduras, Colombia, the Caribbean, and the US.

Their results suggest that ZIKV circulated for long periods of time in these locations before the first infections and associated microcephaly cases were diagnosed — a finding echoed by two other studies out today.

"In each of the regions we could analyze, Zika virus circulated undetected for many months, up to a year or longer, before the first locally-transmitted cases were reported," MacInnis said. "This means the outbreak in these regions was underway much earlier than previously thought."

Using multiplex PCR amplification and/or hybrid capture followed by Illumina HiSeq 2500 or MiSeq paired-end sequencing, members of the team attempted to perform whole-genome sequencing on ZIKV isolates from 221 confirmed or suspected clinical case samples and eight mosquito samples prior to sequencing and assembly. In the process, they put together complete or partial genome sequences for 110 of the ZIKV isolates, using negative control sample sequences to subtract potential contaminant or artifact sequences.

The team's phylogenetic analysis, which included the newly sequenced genomes and more than five-dozen published ZIKV sequences, suggested that the ZIKV strains involved in the recent outbreak share a common ancestor stretching back to early 2014 in Brazil. From there, the outbreak-related virus appears to have spread quickly to other sites in the region and beyond, subsequently forming clusters of genetically-identifiable isolates in the regions affected.

While strains in Brazil and Colombia were similar to one another, for example, all but one of the isolates considered from the continental US fell in a cluster with ZIKVs from Jamaica, the Dominican Republic, and Haiti. Another cluster contained isolates obtained in Honduras, Guatemala, and El Salvador.

In most locales, ZIKV arrival appears to have occurred a few to several months before the first local cases were reported. From the 1,030 mutations detected in the collection, the team narrowed in on potential adaptive mutations that had risen to relatively high frequency in ZIKV isolates and explored potential consequences of viral evolution on available diagnostic targets.

"We expect these data will be useful for researchers designing up to date molecular diagnostics and vaccines and to improve the tools we have to protect and prevent future Zika outbreaks," MacInnis said.

For another study, an international team of investigators sequenced 54 ZIKV isolates, focusing largely on samples collected by a mobile genomics lab that rolled through five Brazilian states over the course of a month in 2016. Many of the samples came from northeastern Brazil, where many ZIKV infections and microcephaly cases have been reported.

Using Oxford Nanopore Technologies MinIon instruments, the researchers did rapid viral genome sequencing on a subset of samples that tested positive for ZIKV by RT-quantitative PCR, generating three-dozen complete or partial genomes for ZIKV isolates from Brazil's northeastern, southeastern, and northern regions, as well as with nine ZIKV genomes from the Rio de Janeiro area, and nine more isolates from Mexico or Colombia.

When they considered these sequences alongside sequences for hundreds more ZIKVs in two phylogeographic models, the researchers saw strain relationships supporting the notion that the ZIKV strain behind the current outbreak can be traced back to early 2014 in Brazil's northeast, well before the outbreak was detected.

"The establishment of the epidemic, the outbreak, in the Americas almost certainly happened in northeast Brazil. Northeast Brazil is the region with the highest number of recorded Zika cases and the highest number of confirmed microcephaly cases," co-corresponding Oliver Pybus, a zoology researcher affiliated with the University of Oxford and the San Francisco-based biotechnology company Metabiota, said during the press briefing. "Our reconstruction shows that there would have been multiple exports of the virus from northeast Brazil to the large, urban regions in southeast Brazil, particularly Sao Paulo and Rio de Janeiro, and then multiple exports to other regions in the Americas."

University of Birmingham researcher Nicolas Loman, a co-corresponding author on that study, led a team that provided more details on the techniques used to directly sequence the ZIKV isolates collected by the mobile lab for a related paper appearing in Nature Protocols.

For the third Zika genomics study published in Nature today, researchers from the Scripps Research Institute, the US Army Medical Research Institute of Infectious Diseases, and elsewhere directly sequenced the genomes of 39 ZIKV isolates from Florida, where local transmission has been reported since last July.

Based on new sequence data for 29 clinical isolates from locally-acquired Zika cases, three isolates from travel-related infections, and seven ZIKV isolates from mosquitoes — together with 65 published ZIKV genome sequences — the team estimated that the virus has been introduced to the state anywhere from four to 40 times.

"It's not a one-off event. This is something that keeps happening over and over again," co-corresponding Kristian Andersen, an immunology and microbial science researcher at the Scripps Research Institute and the Scripps Translational Science Institute, told reporters in the briefing.

Andersen noted that those introductions appear to have started a few months before infections were reported, with many of the strains making their way to Florida from the Caribbean. The region appears to have been hit by locally transmitted infections due to its climate, which is hospitable to Aedes aegypti mosquitoes year-round, coupled with travel from Miami to locations where ZIKV is currently endemic — conditions that exist in a limited number of US locations.  

The newly available genomes are also expected to help in distinguishing between locally transmitted and imported infections more broadly, so mosquito vectors can be targeted in places experiencing local transmission.