NEW YORK – In an effort to get a clearer picture of the spread of highly pathogenic avian influenza H5N1 in the US, a new federal initiative is mandating surveillance testing of bulk milk samples.
In a joint conference with the media on Wednesday, representatives from the US Department of Agriculture, the US Centers for Disease Control and Prevention, and other federal agencies said the new surveillance is a step toward eliminating the B3.13 genotype that began infecting dairy cows in March. However, preliminary sequencing results suggest a severe human infection detected through routine flu surveillance in Louisiana last week is the first such case caused by the more "avian" genotype D1.1, they also said.
Also on Wednesday, California declared a state of emergency for H5N1. With 985 dairies currently undergoing surveillance testing, 641 confirmed positive since August, and 614 dairies still under quarantine all in an attempt to contain the disease to Central California, last week the state detected H5N1 at four dairies in Southern California.
Expansion of nonhuman surveillance efforts comes as evidence accumulates for lethal H5N1 brain infections among nonhuman mammals and recently published research suggesting that a single mutation in the clade 2.3.4.4b H5N1, which includes both B3.13 and D1.1, could increase the virus's infectivity for humans.
Despite these developments, the H5N1 strains causing outbreaks among wild and domestic birds and mammals — and 61 people in the US to date — are currently considered low risk to the public, representatives said at the news conference.
The National Milk Testing Strategy (NMTS) began this week across 13 states representing half of the national milk supply — California, Colorado, Indiana, Maryland, Michigan, Mississippi, Montana, New York, Ohio, Oregon, Pennsylvania, Vermont, and Washington — Eric Deeble, deputy undersecretary for marketing and regulatory programs at the USDA, said on the call.
The milk surveillance program will expand to all 48 contiguous states following the formation of state-by-state cooperative agreements regarding funding and staff, Deeble said. To date, the USDA has also provided $51 million in support to dairy farmers whose herds are quarantined after positive H5N1 testing.
It is unclear which test or tests the state veterinary and public health labs will use for bulk milk surveillance. A recent protocol trialed in Massachusetts and posted to MedRxiv found RT-qPCR and digital PCR approaches to meet slightly different needs.
Sourcing control materials during the first months of the SARS-CoV-2 pandemic was a challenge, and there have also been concerns in the lab community about sourcing H5N1 positive control materials for clinical tests. However, the milk testing protocol was able to use H5N1 virus obtained from pasteurized milk sold in grocery stores.
David O'Connor, a virologist at the University of Wisconsin-Madison and coauthor of the study, said his team tested several methods for isolating viral RNA from store-bought milk and quickly found H5 by PCR. He then designed short amplicon primers using a freely available tool called PrimalScheme but noted that the team is now working to improve the primers further.
Recent studies
The current outbreak among US dairy herds is attributed to the B3.13 genotype of clade 2.3.4.4b H5N1, with samples from animals and infected humans containing small differences named after the infected species and location.
In October, a study in Nature demonstrated that clade 2.3.4.4b H5N1 virus isolated from the eyes of a dairy farm worker with conjunctivitis — dubbed influenza A/Texas/37/2024, or huTX37-H5N1 — could be transmitted through respiratory droplets in lab animals.
Unlike dairy cattle isolates, huTX37 carries an E627K substitution in viral polymerase that can allow avian influenza viruses to efficiently replicate in mammals. When researchers infected lab animals with huTX37-H5N1 via intranasal exposures, the virus spread systemically over a few days. The highest doses of virus caused death in four to six days in mice and ferrets, while the lowest dose took up to 13 days.
The ferret model also showed the strain was transmissible between animals via respiratory droplets regardless of the initial dose of virus administered, although the researchers concluded that the secondary infections were slightly less lethal since only five out of six of the animals died.
The investigators speculated that similar human infections in the US to date may have been mild because they occurred via an ocular route, through splashes of infected milk or poultry feather particles suspended in the air.
"Every effort should be made to contain HPAI H5N1 outbreaks in dairy cattle to limit the possibility of further human infections," they concluded.
Meanwhile, last week a team at Scripps published in Science that one additional mutation in huTX37 — Q226L — was enough to switch the specificity of H5N1 hemagglutinin from bovine to human receptors.
Neurotropism
Although brain infections with HPAI among mammals have been reported since the early 2000s, the severity of infection appears to be increasing across a variety of animals in the past three years of the H5N1 crisis. A handful of SNPs also seem to be driving neurotropism in mammals.
This is especially problematic since "each passage through a mammal is a chance to mutate to more efficiently infect people," Richard Webby, a virologist at St. Jude Children's Research Hospital, said in an interview.
Webby and his colleagues study flu at St. Jude's in part to keep a step ahead in protecting kids with cancer with weakened immune systems. Along with his collaborators, he recently published an algorithm to help triage flu A samples from different host species and environments.
He also recently described the first case of H5N1 infection in a dolphin, which was also the first known case of a dolphin infected with influenza.
"There was a lot of the brain and not so much in the lung," he said.
The virus has also been found in the brains of infected ferrets in the lab as well as red foxes in Germany, Dutch and Canadian carnivores and other wild predators. Last week, the virus killed a cheetah, a mountain lion, a swamphen, a kookaburra and an Indian goose at a zoo in Arizona.
And, neurotropism was also recently reported in naturally infected domestic cats.
However, cows and humans haven't yet shown any evidence of neuropathogenicity, Webby said, although this may be related to current infections that have udder-to-udder and ocular modes of transmission, respectively.
Human surveillance and clinical MDx
Molecular surveillance of humans is also ongoing through wastewater testing at the CDC's National Wastewater Surveillance System. In collaboration with NWSS, WastewaterScan and Verily reported earlier this year that they had developed an H5 assay and discovered the virus in sewage near agricultural processing facilities.
The CDC's influenza surveillance system, meanwhile, has surveilled 65,000 samples this year, Demetre Daskalakis, director of the CDC's National Center for Immunization and Respiratory Diseases, said on Wednesday's call. The agency is also monitoring 9,000 people who are routinely exposed to potentially infected animals.
The surveillance protocol involves clinical testing for flu A followed by subtyping. If a sample were to test negative for A/H1 and A/H3, it would be considered a "presumptive positive" H5 test. This and similar subtyping then requires the sample to be sent to the CDC for confirmatory testing and sequencing.
"It would be nice if more diagnostic tests performed flu subtyping for H1 or H3, which would make identifying H5s easier," said Alex Greninger, a virologist at the University of Washington. However, "doing so uses up channels and doesn't affect clinical care," so not many developers use this approach, he added.
The CDC manufactures the only H5N1 test cleared by the US Food and Drug Administration to date. Granted 510(k) clearance in 2019, the primers of that test have been patented, Greninger noted, which is a concern considering there are no open clinical protocols available online.
A review in the Journal of Clinical Virology published in October noted that the CDC's 510(k)-cleared H5N1 assay might be challenging to use at scale due to issues with the test's design, validation across sample types, manufacturing, and reporting.
Since then, the CDC licensed its test to five reference labs, and on Wednesday, Hologic also announced that it signed an agreement with the CDC to develop new assay reagents. And, a research group published a dual-target H5 subtyping assay.
A representative at the FDA, meanwhile, said that there is currently no relevant declaration under which the FDA may issue emergency use authorizations for H5N1 assays, but also confirmed that the agency doesn't generally expect high-complexity CLIA labs to request marketing authorization to perform HPAI lab-developed tests prior to offering them.
Overall, ramping up surveillance seems to be the best chance at getting ahead of a potential pandemic, but clinical diagnostics may still soon be needed.
While St. Jude's Webby believes that H5N1 is still very much a bird virus, by and large, even after being passaged through cows, an opportunity for "the equivalent of the virologic stars to align" arises each time the virus battles the immune system of any nonhuman mammal.
"Right now, we're giving the virus way too many opportunities," he said.