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

Questions:

* 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.

Astroviruses

* 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!

Questions:

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!

Vignette:

* 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.