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Sequencing Studies Track Dengue Virus Genetics Across Transmission Stages

NEW YORK (GenomeWeb) – Studies published online this week in PLOS Neglected Tropical Diseases are helping to untangle the genetic diversity and genetic limits of dengue viruses found in human hosts or mosquito vectors that transmit them.

For the first of these, researchers from Singapore, Vietnam, the UK, and Australia did whole-genome sequencing on dengue viruses in the blood of a dozen infected individuals and three mosquitoes per person that were fed infected blood from the patients.

The team saw a dramatic drop in genetic diversity as the virus moved from human hosts to mosquitoes and from mosquito abdomens to salivary glands, with more than 90 percent of the original SNPs disappearing. Nevertheless, the virus' overall genetic diversity remained steady as new single nucleotide changes appeared at each transmission stage.

"These matched samples allowed us to estimate the size of the population drop that occurs during establishment of infection in the mosquito, track changes in viral intra-host variant repertoires at different stages in transmission, and investigate the possibility of host-specific immune selection pressures acting on the virus population," senior author Martin Hibberd, an infectious disease researcher at the Genome Institute of Singapore, and his colleagues wrote.

Humans become infected with dengue virus through bites from Aedes aegypti or Ae. albopictus mosquitoes with dengue virus in their salivary glands. Conversely, the dengue virus re-enters mosquitoes that bite infected humans, replicating in the gut before moving to mosquito salivary glands and other organs.

The research groups reasoned that by untangling population changes that occur in dengue virus during these transitions, they might be able to find clues to thwarting or treating human infections.

Using PCR amplification and Illumina HiSeq sequencing, Hibberd's team and collaborators at the Genome Institute of Singapore generated whole genome sequences for dengue virus 2 isolates from 12 human blood samples, 21 matched samples from Ae. aegypti mosquito abdomens, and 25 matched mosquito salivary gland samples.

The team's variant analyses indicated that the level of within-individual genetic diversity was comparable across each of the human or mosquito samples tested.

But this diversity belied the significant single nucleotide variant changes that occurred as dengue virus moved from human blood to mosquito abdomen to mosquito salivary gland sites: fewer than 10 percent of SNPs survived the move from human blood to the mosquito abdomen, on average, and not quite 8 percent of variants in viruses from the mosquito abdomen made it to the salivary gland.

Rather, random mutations arising in each new locale seemed to maintain dengue virus diversity, the researchers explained, with selective pressures in human and mosquito hosts partly dictating the nature of these alterations.

Meanwhile, in the second study, a Singapore team that included some of the same investigators compared viral genome patterns in blood samples from early and late stages of dengue virus 1 infection in 12 individuals as well as sequences from paired Ae. aegypti or Ae. albopictus mosquito samples to search for parts of the dengue virus 1 genome that tend to resist mutation during transmission.

"Deep sequencing of the dengue virus 1 genome directly from the blood of twelve dengue patients and from mosquitoes given this blood showed consistent and distinct mutation patterns during infection," senior author Eng Eong Ooi, an emerging infectious diseases researcher with the Duke-NUS Graduate Medical School in Singapore, and colleagues explained.

For example, results from that study, which relied on Illumina and ABI SOLiD sequencing, suggest that a portion of the dengue virus 1 genome that codes for the non-structural protein NS3 is genetically constrained and impervious to mutation. The team also picked up on slightly different dengue virus mutation patterns during passage into each mosquito vector species.