Last January, a team of researchers searching for the coronavirus in New York City’s wastewater spotted something strange in their samples. The viral fragments they found had a unique constellation of mutations that had never been reported before in human patients — a potential sign of a new, previously undetected variant.

For the past year, these oddball sequences, or what the scientists call “cryptic lineages,” have continued to pop up in the city’s wastewater.

There is no evidence that the lineages, which have been circulating for at least a year without overtaking delta or omicron, pose an elevated health risk to humans. But the researchers, whose findings were published in Nature Communications on Thursday, still have no idea where they came from.

“At this point, what we can say is that we haven’t found the cryptic lineages in human databases, and we have looked all over,” said Monica Trujillo, a microbiologist at Queensborough Community College and an author of the new paper.

The researchers themselves are torn about the lineages’ origins. Some lean toward the explanation that the virus is coming from people whose infections aren’t being captured by sequencing. But others suspect that the lineages may be coming from virus-infected animals, possibly the city’s enormous population of rats. Even then, the favored theory can change from day-to-day or hour-to-hour.

Answers remain elusive. “I think it’s really important that we find the source, and we have not been able to pin that down,” said John Dennehy, a virologist at Queens College and an author of the paper.

Strange Sequences

The researchers — who also include Marc Johnson, a virologist at the University of Missouri; Davida Smyth, a microbiologist at Texas A&M University and others — have been sampling wastewater from 14 treatment plants in New York City since June 2020. In January of 2021, they began doing targeted sequencing of the samples, focusing on part of the gene for the virus’s all-important spike protein.

Although this approach provides a limited look at the viral genome, it allows researchers to extract a lot of data from wastewater, in which the virus is typically fragmented. Viral fragments with novel patterns of mutations appeared repeatedly at a handful of treatment plants, the researchers found. (They could not disclose the specific plants or areas of the city, they said.)

“To date we have not seen these variants among clinical patients in NYC,” said Michael Lanza, a spokesperson for New York City Department of Health and Mental Hygiene.

Researchers at the University of California, Berkeley, have found similar sequences in one California sewershed, said Rose Kantor, a microbiologist at the university.

The scientists’ continuing quest to figure out where the sequences are coming from highlights both the potential of wastewater surveillance, which can help scientists keep tabs on how the virus is evolving, and the challenge of making sense of any anomalies pulled out of the murk.

“We really struggled trying to understand what it was that we had,” Trujillo said.

The lineages could be coming from people whose infections have escaped detection or whose virus has not been sequenced. But the fact that they kept turning up at the same few wastewater plants makes this theory less likely, the researchers said, given that New Yorkers, and any variants they may be carrying, tend to move throughout the city without restriction.

Still, Dennehy speculated that the sequences could be coming from people who are confined to long-term health care facilities in just a few areas of the city. But he has not been able to prove it.

“We were able to pin it down to a very small area of the sewershed,” Dennehy said. “And I emailed doctors and hospitals in those areas and never once got a response to my emails.”

Indeed, people who have compromised immune systems may have more difficulty fighting off the virus, giving it more opportunities to mutate. Many scientists theorize that omicron emerged from an immunocompromised patient.

Intriguingly, some of the cryptic lineages have some of the same mutations as omicron, or mutations in the same locations. Laboratory experiments suggest that these lineages may also be able to evade some antibodies.

The New York City lineages might be a result of the same kind of selective pressure to evade some of the body’s immune defenses, the researchers theorize.

An Animal Origin?

On the other hand, the lineages have been circulating for long enough now that they should have appeared in at least one sample sequenced from an infected person, some scientists said.

“To have something in a sewershed that you’re detecting, you need a fair bit of it around,” said Dr. Adam Lauring, a virologist at the University of Michigan, who was not involved in the research.

Johnson, the Missouri virologist, agrees. He favors the hypothesis that the sequences are coming from animals, perhaps a few specific populations with limited territories. In May and June of 2021, when the number of human COVID-19 cases in the city was low, the mysterious lineages made up a greater proportion of the viral RNA in wastewater, suggesting that they may have come from a nonhuman source.

The researchers initially considered a diverse array of potential hosts, from squirrels to skunks. “This is a very promiscuous virus,” Johnson said. “It can infect all kinds of species.”

To narrow down the possibilities, they went back to the wastewater, assuming that any animal that was shedding virus might be leaving its own genetic material behind, too.

Although a vast majority of the genetic material in the water came from humans, small amounts of RNA from dogs, cats and rats were also present, the scientists found.

Johnson has been considering rats, which roam the city by the millions. In his lab, he created pseudoviruses — harmless, nonreplicating viruses — with the same mutations present in the cryptic sequences. The pseudoviruses were able to infect both mouse and rat cells, he found. The original version of the virus does not appear able to infect rodents, although some other variants, like beta, can.

“So in and of itself, that isn’t huge data, but it is at least consistent with the idea that it’s coming from rodents,” Johnson said.

Since last summer, the scientists have been working with Animal and Plant Health Inspection Service at the U.S. Department of Agriculture to look for signs of the virus in blood and fecal samples from local rats. So far, they’ve come up empty.

“Maybe we’re not hitting the right animals,” Dennehy said.

Or maybe rats aren’t the source of the mystery lineages. Scientists have repeatedly found that human can pass the virus to animals, especially pets, zoo animals, farmed mink and others with which they are in frequent contact. That has raised concerns that the virus might establish itself in an animal reservoir, where it might mutate and get passed back to humans.

But rats have not typically been high on the list of concern, and there has not been any evidence that the virus is circulating in wild rats. The pathway by which humans could have infected rats is also unknown.

“Nothing makes perfect sense,” Johnson said.

But some kind of animal origin remains a possibility, scientists said.

“It’s just as plausible, if not more plausible, than a human origin,” Lauring said.

So the search continues. Johnson has developed a new technique that can amplify only non-omicron sequences, which should make it easier to detect the lineages. He has also begun searching for similar lineages in sewage samples from other states, which might help provide further clues to their origins.

“We will know eventually,” Johnson said.

This article originally appeared in The New York Times.

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