By Kai Kupferschmidt
The avian influenza virus that has been infecting dairy cows and spreading alarm in the United States was expected to reach Germany this week. But that’s actually good news. A shipment of samples of the H5N1 virus from Cornell University virologist Diego Diel is destined for the Federal Research Institute for Animal Health in Riems, which has one of the rare high-security labs worldwide that are equipped to handle such dangerous pathogens in cattle and other large animals. There, veterinarian Martin Beer will use the samples to infect dairy cows, in search of a fuller picture of the threat the virus poses, to both cattle and people, than researchers have been able to glean from spotty data collected in the field.
Six weeks into the outbreak that has spread to farms in nine U.S. states, the flow of data from those locations remains limited as public health officials sort out authorities and some farms resist oversight. “It’s incredibly difficult to get the right sample sets off the infected farms,” says Richard Webby, an avian influenza researcher at St. Jude Children’s Research Hospital. “It’s clearly a barrier to understanding what’s going on. … That’s why these experimental infections of cows are really going to be super informative.”
Beer and other scientists at biosafety level-3 (BSL-3) labs that handle large animals hope to glean clues about how to head off a dire scenario in which the virus establishes itself in cows across the United States, or eventually worldwide. That could harm the beef and dairy industries and increase the risk of a human pandemic. “Nobody wants this dangerous virus to become entrenched in a new species that we use to produce food and that has so much close contact to humans,” Beer says.
As he and other groups set up their controlled infection experiments in cattle, a preprint posted last week gives a possible explanation for why the virus seems to thrive in the cow udder—and therefore ends up in milk. Influenza viruses bind well to certain cell surface carbohydrates called sialic acids, which vary by species. Avian viruses latch onto duck and chicken sialic acids, whereas human influenza viruses latch onto certain mammalian sialic acids. In the preprint, posted on bioRxiv, researchers from Denmark found duck-type as well as human-type receptors in the mammary gland of a dairy cow, which could make it a natural target for the bird virus.
The results are potentially important but also preliminary, Beer cautions, noting that distinguishing between some types of sialic acids can be difficult. But Webby, one of the preprint’s authors, says it’s obvious the udders are especially hospitable to the virus. “We clearly know the receptors are in there for the avian virus because it's growing like a weed.” The authors also expressed concern that cows, like pigs, could become influenza “mixing vessels” that create dangerous new human strains when avian and mammalian flu viruses that simultaneously infect an animal exchange genes. Beer says he’s more worried, however, about an avian virus slowly adapting.
So far, the threat to people has been limited. The only confirmed case to date, a Texas dairy worker who reported close contact with milked cows on an affected farm, developed conjunctivitis, but his symptoms resolved after he was given the antiflu drug oseltamivir. An eye swab yielded virus of the same H5N1 genotype, called B3.13, that has been infecting cows, researchers reported last week in The New England Journal of Medicine.
Human eyes may be uniquely vulnerable to the strain because they contain the same H5N1 receptor found in ducks and cow udders. But the rapid viral growth in cow udders raises concerns that the avian virus could learn to latch on to the mammal-specific virus receptor also in the udder, says Tom Peacock, an influenza virologist at the Pirbright Institute. “It seems a good reason to get this eradicated from cattle as soon as possible.”
One goal of the lab work is to investigate how the virus is spreading from cow to cow. The working theory is that lactating cows with infected udders are spreading the virus to other dairy cows during the milking process, but that’s based on circumstantial evidence such as the presence of H5N1 in environmental samples from milk parlors. “The big question right now is whether the virus is mechanically transmitted or can be transmitted from cow to cow via aerosol as well,” says Jürgen Richt, a virologist at Kansas State University.
The best way to test this is in well-designed animal experiments in highly secure labs, he says. “There are only a few facilities that can do this kind of work, so we have been coordinating with each other who is doing what.”
Richt, who runs one such facility, plans to inoculate the nose and mouth of male and female cattle with the same strain Diel sent Beer. “Then we will look at what’s happening within the animals,” he says. “We will kill some of them after 4 or 5 days and look where the virus is in the body.” Others will be watched for weeks to see whether they develop antibodies to H5N1 and how long they shed the virus in various ways. And 2 days after some of these initial infections, new cows, called sentinels, will be added to the mix to see whether they can catch the virus.
While Richt is doing these experiments with nonlactating cows, Beer will work with lactating cows, depositing the virus straight inside all four of the animals’ teats. And Volker Gerdts, director of the Vaccine and Infectious Disease Organization in Saskatchewan in Canada, is planning similar experiments with calves in the organization’s BSL-3 facility.
Using lactating animals adds a whole layer of complexity, because dairy cows are very sensitive to changes in their conditions and because they need to be regularly milked, Beer says. “We just bought three new milking machines to take into the BSL-3.” But these kinds of experiments are “absolutely crucial,” says evolutionary biologist Michael Worobey of the University of Arizona. “It’s still possible that this will become an H5N1 pandemic. And those experiments will help understand if that’s likely or less likely.” (Beer previously infected cattle in 2007 with a different form of H5N1.)
Other researchers are studying the cow flu strain in cell culture. Diel, for instance, is examining how well the virus replicates in different cell types. He also wants to see whether the affinity of the bovine H5N1’s hemagglutinin, the surface protein the virus uses to bind to cells, has changed to one better suited for the sialic acids on cells in the human respiratory tract, a sign that the virus is adapting to transmission in mammals.
Some scientists are anxiously looking for signs of the bird flu becoming human-adapted by tracking the mutations seen in the viral sequences from infected cows. A few, such as one called 631L, have already been found and appear to make H5N1’s polymerase, the enzyme the virus makes to copy its genome, work better in mammals. But similar mutations have been seen in H5N1 cases in other animals, and there has been little other evidence of mammalian adaption so far, Peacock says.
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