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Viral Metagenomics Research Hints at Potential Ecological Influence of Treated Waste

BOSTON (GenomeWeb News) – Human feces, sewage, and even reclaimed wastewater contain a host of viruses — many uncharacterized — that could ultimately affect other organisms in our environment, new research is revealing.
A team of researchers, led by University of South Florida marine microbiologist Mya Breitbart, have uncovered unexpected viruses — among them plant and insect viruses — in human fecal samples and in treated and untreated sewage. The work provides clues about viruses that could ultimately become bioindicators, flagging areas contaminated by human sewage. It may also have ecological implications, since re-cycled wastewater is increasingly being used as a source of water for irrigation.
Karyna Rosario, a graduate student in Breitbart’s lab presented the work at the American Society for Microbiology meeting yesterday.
Although it’s possible to estimate the overall viral abundance in the environment, Rosario explained, identifying specific viruses is trickier. Metagenomics offers a potential solution, making it possible to look at viral diversity and discover viruses that would otherwise remain imperceptible.
Because viruses in the human gastrointestinal tract can influence human body functions and the microbial flora dwelling within, Breitbart and her team decided to explore this viral diversity. They first applied viral metagenomics to human fecal samples, extracting viral nucleic acids — either DNA or RNA — from filtered and purified human fecal samples. By sequencing these nucleic acids and comparing the sequences to those in available databases, they were able to start characterizing the viral flora in the feces of healthy humans.
The researchers found that some 60 percent of the DNA viral sequence was from unknown viruses. “There’s a lot of novelty in these genes and we have no clue what these sequences are,” Rosario said. Amongst the known genes, most sequences identified were from phages — viruses that infect bacteria.
While that made sense, Rosario said, given the abundance of bacteria in the human gut, the team still expected to find human viruses when the looked at the RNA virus metagenome. Instead, the viral sequences most often came from a plant pathogen called the pepper mild mottle virus or PMMoV, which causes malformation and mottling in pepper plants.
They found PMMoV in three different libraries, all of which were sequenced with high-throughput pyrosequencing by collaborators at the Genome Institute of Singapore: two libraries of RNA viral genes from one individual’s feces at different times and a third library from another individual.
And the pepper virus wasn’t just detected in those two individuals. When the researchers expanded their search to include 18 people from San Diego or Singapore, they found PMMoV sequence in the feces of 78 percent of those living in San Diego and 67 percent of those living in Singapore. It was also detected in every raw sewage sample tested in 11 states.
“The first guess is that it’s probably coming from our food,” Rosario said.
But, as it turned out, those carrying PMMoV weren’t necessarily scarfing down peppers. When Breitbart and her team tested a variety of foods for PMMoV, they didn’t find the virus in any of the fresh peppers tested. They did find it in several processed foods, though, including chili sauces, chili powder, and Indian curry.
Subsequent experiments indicated that the PMMoV that had passed through the human digestive system could successfully infect plants, suggesting humans unwittingly help the viruses get to their preferred hosts. “We are probably vectors of the virus,” Rosario said.
That made the researchers wonder what happens to the viruses in human waste when we treat sewage and convert it back into water — a process called wastewater reclamation. Because of water shortages in some areas, reclaimed water has increasingly become an alternative to other water sources for agricultural and other irrigation.
Breitbart’s team decided to use their metagenomic approach, combined with transmission electron microscopy and PCR analysis, to see whether DNA or RNA viruses from human waste survived sewage treatment. So far they have discovered an abundance of viruses in the treated wastewater, including PMMoV and many unknown viral sequences.
Once again, there were surprises amongst the known genes. The RNA virus metagenome boasted a bounty of sequences from viruses that normally infect insects.
The team is continuing their characterization of these and other viruses in the reclaimed wastewater — work that may inform the way such water is used in the future. And, Rosario said, by combining metagenomics with complementary techniques, they hope to get a more complete understanding of what’s happening in that water source.
“Metagenomic sequencing can reveal the unexpected,” Rosario concluded. “It shows you what’s there, not just what you’re looking for.”

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