A team of researchers looking for the coronavirus in New York City’s wastewater found something unusual in their samples last January. The viral pieces they discovered exhibited a unique set of mutations that had never been seen in human patients before, suggesting the presence of a new, previously unreported strain.

“Cryptic lineages,” as they are called, have been popping up again and again in the city’s wastewater for the last year or so.

This means there is no proof that the lineages that have been around for at least a year without taking over delta or omicron are more dangerous to humans. However, the researchers, whose findings were published in Nature Communications today, are still unsure 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,” says Monica Trujillo, the author of the new study.

The researchers are split on the origins of the lineages. Some speculate that the virus is being spread by persons whose infections aren’t being detected by sequencing. Others, however, believe the lineages are derived from virus-infected animals, such as the city’s massive rat population. Even so, the most popular hypothesis can shift from day to day or hour to hour.

No answers yet.

“I think it’s really important that we find the source, and we have not been able to pin that down,” adds John Dennehy, one of the authors of the paper.

Since June 2020, the team — which includes Marc Johnson, Davida Smyth, and others — has been sampling wastewater from 14 treatment plants in New York City.

They began focused sequencing of the samples in January 2021, focusing on a portion of the gene for the virus’s crucial spike protein.

Although this method only gives researchers a limited view of the viral genome, it does allow them to retrieve a lot of data from wastewater, where the virus is usually fragmented. The researchers discovered that viral pieces with unique mutation patterns emerged regularly at a number of treated plants. (They indicated they couldn’t reveal the specific plants or locations of the city.)

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

According to Rose Kantor, a microbiologist at the University of California, Berkeley, researchers discovered similar sequences in one California sewershed.

The scientists’ ongoing effort to figure out where the sequences came from illustrates both the potential of wastewater surveillance, which can help scientists track how the virus evolves, and the difficulty of making sense of any abnormalities extracted from the muck.

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

The lineages could be from persons who have had their infections go undetected or whose virus has not been sequenced. The fact that they kept showing up at the same few wastewater plants, however, makes this explanation less feasible, according to the researchers, because New Yorkers, and whatever variants they may be carrying, are free to roam across the city.

Nonetheless, Dennehy theorized that the sequences might be originating from persons confined to long-term health care facilities in a few sections of the city. He hasn’t been able to prove it, though.

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

Indeed, people with weakened immune systems may have a harder time battling the virus, giving it more chances to mutate. Omicron is thought to have originated in an immunocompromised patient, according to many scientists.

Surprisingly, several cryptic lineages share some of omicron’s mutations or mutations in similar sites. Experiments in the lab suggest that these lineages may be able to dodge some antibodies as well.

The researchers hypothesize that the New York City lineages are the consequence of the same kind of selective pressure to elude certain of the body’s defensive responses.

However, other scientists believe that the lineages have been circulating for long enough that they should have surfaced in at least one sample sequenced from an infected person.

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

Johnson, a virologist from Missouri, agrees. He supports the idea that the sequences come from animals, possibly from a few small groups with limited territory. When the number of human COVID-19 cases in the city was low in May and June of 2021, the unknown lineages made up a larger proportion of the viral RNA in wastewater, implying that they may have originated from a nonhuman source.

Initially, the researchers explored a wide range of potential hosts, including squirrels and skunks.

“This is a very promiscuous virus,” Johnson adds. “It can infect all kinds of species.”

They went back to the wastewater to narrow down the options, presuming that any animal that was shedding virus was also leaving its own genetic material behind.

Although humans accounted for the vast majority of the genetic material in the water, the scientists discovered that minor amounts of RNA from dogs, cats, and rodents were also present.

Johnson has considered the millions of rats that swarm the metropolis. He generated pseudoviruses in his lab, which are harmless, nonreplicating viruses with the same mutations as the cryptic sequences. He discovered that the pseudoviruses could infect both mouse and rat cells. Although the original version of the virus does not appear to be capable of infecting rats, other variations, such as beta, do.

“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,” according to Johnson.

Since last summer, the scientists have been collaborating with the US Department of Agriculture’s Animal and Plant Health Inspection Service to seek for symptoms of the virus in blood and fecal samples from local rats. They’ve come up empty so far.

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

Maybe the unknown lineages aren’t caused by rats. Scientists have discovered that humans can spread the virus on to animals, including pets, zoo animals, farmed mink, and others with whom they come into contact on a regular basis. Concerns have been expressed that the virus could establish itself in an animal reservoir, where it could mutate and spread to people.

However, rats have never been a major source of concern, and there has been little evidence that the virus is spreading among wild rats. The mechanism by which rats became infected by humans is likewise uncertain.

“Nothing makes perfect sense,” Johnson adds.

However, scientists believe that an animal origin is still a possibility.

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

So the search goes on. Johnson has created a new approach that can only amplify non-omicron sequences, making lineage detection easier. He’s also started looking for comparable lineages in sewage samples from other states, which could lead to more information about their origins.

“We will know eventually,” Johnson says.

Source: 10.1038/s41467-022-28246-3

Image Credit: Getty

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