Skip to main content
Premium Trial:

Request an Annual Quote

Verily Team Finds H5 Avian Flu Subtype in Wastewater as Expanded CDC Surveillance Plan Takes Shape

Avian Flu

This story has been edited to correct information about the study as well as the target of the Verily assay. It is the H5 subtype of influenza A, not the H5N1 strain specifically. 

NEW YORK – Scientists at Verily, an Alphabet health technology company, in collaboration with academic program WastewaterSCAN, recently developed a digital PCR method to detect the H5 subtype of influenza A in wastewater and subsequently discovered the presence of H5 in samples collected in Texas. The detection appears to be linked to highly pathogenic H5N1 circulating among local dairy cows. 

Although the work was not part of the firm's contract with the US Centers for Disease Control and Prevention, the CDC's surveillance strategies are anticipated to soon include surveilling flu A generally rather than the H5 subtype specifically.

Also known as bird flu, the H5N1 strain of avian influenza (HPAI) virus prefers wild and domestic bird hosts. In human hosts, infection is usually limited to poultry farmers, while spread among other mammals is typically confined to zoos, fur farms, or herds of sea lions.

Infections among cows in the US first detected in March, however, seem to be taking a different track, with scientists last week reporting evidence of fatal cross-species mammalian infections. Dairy farmers in Texas inadvertently fed a clowder of two dozen farm cats unpasteurized milk from unwell cows, and within three days, half the cats were dead. Both bovine and feline samples later tested positive for H5N1. The US Department of Agriculture website has since confirmed dairy cow infections in nine states.

Verily's H5 wastewater-based epidemiology (WBE) testing coincided with this unusual animal outbreak, and likely detected signals of it.

According to Bradley White, principal scientist at Verily, the team started spotting unseasonal influenza A spikes during routine wastewater monitoring of plants in Texas. The result raised concerns for Verily and WastewaterSCAN, its WBE collaborator.

The Verily team developed an H5-specific digital PCR assay for its Bio-Rad QX600 Droplet Digital PCR systems, validated it on archived samples, then set about testing archived wastewater samples from three treatment plants that had shown the unusual flu A spikes.

As described in a preprint posted to MedRxiv last week, samples from 59 of the 190 US wastewater treatment plants in WastewaterSCAN's nationwide program showed the unseasonal flu A spikes. The team was also able to track levels rising and falling over time and verify that the overall higher influenza A levels were almost completely driven by the H5 subtype in the three 'sentinel' treatment plants in Texas.

With only a single reported human case of H5N1 infection in the area — in a person who worked at a dairy facility — the H5 in wastewater was a conundrum, but the team hypothesizes that the signal is coming from milk and beef processing plants permitted to release treated waste into the sanitary sewer system. Verily and WastewaterSCAN are now undertaking sewershed sampling in manholes and lift stations to localize the source of the virus, White said.

Expanding H5 wastewater surveillance?

Verily serves as lab partner to WastewaterSCAN, a research group codirected by Stanford University's Alexandria Boehm and Emory University's Marlene Wolfe and funded through philanthropy.

Each week, Verily tests samples from approximately 200 sewage treatment plants in 36 states, surveilling the pathogen nucleic acids shed by 42 million people.

In September, Verily won a contract from the CDC — previously awarded to testing firm Biobot Analytics — and is now testing samples from more than 400 US wastewater treatment plants for the CDC's National Wastewater Surveillance System (NWSS) and local public health departments.

The H5 assay wasn't developed for the CDC, White said, and the study described in the preprint was preformed only on samples from the WastewaterSCAN program and does not reflect Verily's CDC NWSS testing.

Marc Johnson, a virologist at the University of Missouri who helped spearhead that state's Sewershed Surveillance Project during the pandemic, has also recently developed several H5 digital PCR assays and is currently using them in the lab.

The virus has been circulating in cattle since approximately last November, Johnson said in an email, and yet it was completely missed until March. 

"It is astounding to me that it got this far unnoticed," he said.

Johnson's lab is currently using the assay to test samples other than wastewater. "I never thought I would be running H5 assays on milk that was purchased at a grocery store, but that is what we are currently doing," he said.

Johnson considers the Verily/WastewaterSCAN preprint to be solid work and said it is also "good to see that there is a wastewater surveillance group that is allowed to be this nimble."

His team currently surveils approximately 60 percent of the state population for the Missouri health department, he said, which in turn contributes data to the CDC and NWSS.

"Public health in general is interested in collecting data that is actionable and can be clearly messaged to the public," he said. So, although academic labs like his were eager to begin surveilling for H5 the moment of the first report, "public health does not move that fast."

Currently, the NWSS public dashboard only publicly reports COVID and mpox.

Amy Kirby, head of the NWSS division, said in an email that some NWSS sites also began collecting data on the presence of influenza A, influenza B, and respiratory syncytial virus during the 2023-2024 respiratory season.

The CDC does not currently have any plans to add H5-specific testing to its wastewater surveillance program, she said, but the agency "is monitoring wastewater data for any evidence of unusual levels of influenza and is working to develop and validate an influenza wastewater metric that will be publicly shared soon" on the CDC's website.

Currently, more than 600 sites within the program are collecting data on flu A, according to a CDC website, and monitoring for unusual levels. Kirby said some sites are also now testing for flu B and respiratory syncytial virus, with still more locales using the data to inform their public health actions.

Kirby said the CDC's NWSS division works with its local partners to help evaluate the use of wastewater data for additional targets in order to assess whether they might be valuable to inform public health actions.

"In addition, some sites are expanding testing to include other targets that may be priorities in their jurisdiction," she said.

Through a collaboration with GT Molecular, NWSS has already supported the development of digital PCR wastewater assays for more than 30 microbial targets, Kirby said, as part of a planned surveillance expansion. These assays are being developed for use on the Bio-Rad systems as well as Qiagen's QiAcuity digital PCR systems, as previously reported.

Kirby said that the GT Molecular tests for SARS-CoV-2, influenza A and B, RSV, and mpox are now available for use, and NWSS will be evaluating the optimal use of the remaining assays to support public health action. Future targets under consideration include West Nile virus, dengue virus, and norovirus, she also said.

University of Missouri's Johnson said that, to his mind, using an H5-specific test would be better than general flu A surveillance since "it is better to know what virus you are detecting."

However, "I'd be willing to bet that H5 will make it on to the [CDC] panel pretty soon. … It's just a question of how soon," he said — assuming this outbreak doesn't fizzle out, which, he said, currently seems unlikely.

Verily's future directions

Currently, WastewaterSCAN performs WBE for 11 infectious disease pathogens and provides the data in a public dashboard. Specifically, the academic program monitors influenza, respiratory syncytial virus, SARS-CoV-2, human metapneumovirus, parainfluenza, norovirus GII, rotavirus, enterovirus D68, mpox, Candida auris, and hepatitis A.

WastewaterSCAN also conducts studies to connect wastewater detection with local epidemiology and publishes the data in peer-reviewed journals.

For example, in February the team showed RSV infection positivity and hospitalization rates have a somewhat complex relationship with RSV RNA concentrations in wastewater solids. Using data from 176 sites collected during the 2022–2023 RSV season, the WastewaterSCAN team found differences in onset and peaks in wastewater versus clinical data that it suggested may relate to unrecognized, mild RSV cases and prolonged viral shedding.

Verily, meanwhile, tests for WastewaterSCAN's 11 targets and provides its services to groups like the California Department of Public Health and others.

White said he and his team use Bio-Rad's QX600 Droplet Digital PCR system for this work because they can multiplex up to nine targets, the 96-well plate format is highly scalable, and the 20,000 droplets it generates provide high sensitivity. 

The Verily and WastewaterSCAN team contributed to a report in the New England Journal of Medicine on detection of mpox in wastewater last year and recently demonstrated dengue virus detection from wastewater in the Miami-Dade County area that correlated with local human infections. In a study published in Environmental Science & Technology Letters late last year, the team revealed the presence of dengue serovar 3 but the absence of other serovars, correlating with local cases.

To White's mind, the limits of wastewater surveillance for viruses remain to be seen. He was skeptical that targets like RSV would be detectable, for example. But "everything we try, shockingly, has actually worked," he said.

The team has also managed to streamline plant onboarding and sample logistics to automate many of the laboratory processes and is now focused on conducting R&D for additional assays and expanding its partnerships with public health in the US and globally.

"Given the ongoing and growing need for this nascent monitoring platform, Verily believes we are well positioned to serve customers in the wastewater field," White said.