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Thursday, February 25, 2010

AGBT 2010 - David Wang, Washington in St. Louis school of Medicine

David Wang, Washington in St. Louis school of Medicine

Metagenomic approaches to Pathogen Discovery

Disease this group studies
* Respiratory tract infections
* Diarrheal diseases

Which viruses cause Respiratory tract infections? Lots!
* 20-30% can not be associated with any known virus.

genomic approach
* Get nucleic acids
* Use ViroChip
* use Mass Sequencing
* now use NGS - 454 + a little Illumina

Lots of this was published - 20,000 sequences on an array... etc

Good example for the application of this information: SARS.
* Collaborated with CDC
* ViroChip identified coronavirus signal
* Elution and sequencing of SARS fragments from microarray (affinity capture-sequencing.)
* Sanger shotgun sequencing of SARS culture -- 80% of SARS genome.
* (Rota et al, Science 2003)

Applications to respiratory infections

Genomic screening for Novel viruses - pre-NGS
* start with 3 year old with pneumonia
* Sample was negative for all platforms at the time... used Sequence Analysis Pipeline (High throughput sanger sequencing.)
* less than 50% identity to known viruses

Polyomavirus background
* circular, double stranded DNA
* infect mammals and birds
* two human viruses of this type was known at the time
* has a T-antigen (may be cancer related)
* pathogenic in compromised individuals
* persistent once contacted.

Novelty of the virus they isolated
* thought they'd found the 3rd polyomavirus
* turned out they HAD found a 3rd... and another one in sweeden (4th) by the same method by another group at the same time.

* prevalence is 1-8%
* 80-90% of adults have antibodies against WUV.
* Infection peaks in early childhood.
* There is still a lot of work left to understand how they work, what they're doing, etc

* can it be cultured?
* is it associated with cancer, respiratory disease, etc?

On to Diarrhea...
* 1.5-2M children die from acute diarrhea each year
* Major viral causes of diarrhea
* not much activity on this now.
* even today 40% of known diarrhea can't be identified as a known virus.

Start with Pediatric
* children had failed known screening.
* 1864 high quality unique sequences - 14 were viral
* as many as 3 viruses in one sample
* evidence for novel viruses.

* ssRNA ~6-8kb in length
* only one species had been described - 8 serotypes

Novel Astroviruses fragments detected.
* MLB1 is clearly a novel astrovirus
* does not cluster with other serotypes
* globally widespread!

1. is it a cause of human diarrhea?
2. does it cause disease elsewhere, but shed in stool?
3. is it commensal/symbiotic part of the virome?
4. is it a result of dietary ingestion?

How do we go deeper into this story?
* pooling and NGS!

* Gastroenteritis outbreak in day care, Virginia
* Found another novel Astrovirus
* ~2000 sequences - 313 novel
* Assembled all of this:
** 6.5 kb contig
* one single run of 454 generated the entire genome, with 5' end missing 5 nt.

* Same questions as before, however.

In new work, have now identified 3 new astroviruses.

What is better than one stool samples? Look at 1000's of them!
[Picture of a field of toilets...]

Working now, with raw sewage samples. Fecal and urine microbiome. Correlate back to individual viruses.



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