Chinese researchers who did extensive work on H7N9 viruses from birds and humans found that one of the human strains was highly transmissible by aerosol droplets in ferrets, fueling more concerns that the new virus could spread between people.
The potential for aerosol spread is one of the key factors health officials use in gauging a new virus's pandemic potential, and the new study follows closely on the heels of two others that also found evidence of respiratory droplet transmission in ferrets.
The newest findings were reported today in an early online edition of Science by a team based at the Harbin Veterinary Research Institute, a World Health Organization (WHO) collaborating center.
Genetic comparisons produce new clues
Scientists ran multiple tests on H7N9 viruses obtained during poultry surveillance and isolated from human cases to get a clearer picture of its pathogenicity, virulence, replication, and transmissibility.
They sequenced the genomes of 37 representative H7N9 samples, most of which came from live-poultry markets, and compared them with five human isolates. The hemagglutinin (HA) and neuraminidase (NA) genes were highly similar, but they found more diversity in the six internal genes. They also found that the viruses are still capable of frequent reassortment and rapid evolution.
When they examined the basic polymerase 2 (PB2) gene for amino acids associated with flu virulence and transmission in mammals, they found that all of the bird and environment samples had the amino acid combination 627E/701D. The human isolates, in contrast, had either the 627K or 701N mutation, both of which are important for virulence and transmission.
The group wrote that the findings suggest the mutations may have occurred during replication in humans.
Experiments to explore receptor binding, another factor that plays a role in flu virus replication and transmission, identified a 1243V mutation that—similar to the Q226L mutation—may play a key role in exclusive binding to humanlike receptors for two of the avian isolates and two of the human samples.
Tests track aerosol transmission, other factors
Virulence and infection tests in birds confirmed that the H7N9 virus was low pathogenic in poultry and that infected chickens shed the virus for up to 7 days.The researchers said this finding suggests that chickens "may be one of the major carriers and spreaders of H7N9 viruses in the live poultry markets."
In tests on mice that were given lethal doses of H7N9, no signs of disease or death were seen in the ones that received viruses isolated from birds, but the animals infected with human isolates lost weight, got very sick, or died. Similarly, the group's replication tests on mice infected with human strains found higher viral titers in the nasal passages and lungs when compared with animals infected by the bird strains.
Replication experiments in ferrets also showed differences between the bird and human H7N9 strains. The group's pathology tests on ferret lung samples found severe bronchopneumonia and prominent viral antigen expression in the animals infected with three human strains and one of the bird strains. Ferret lungs, though, appeared normal after infection with a poultry H7N9 strain.
Aerosol transmission studies involved placing uninfected ferrets in cages adjacent to those housing infected ferrets. The investigators found H7N9 in one ferret exposed to those infected with one of the bird strains and two human strains isolated from some of the first patients in Shanghai. However, the virus was detected in all three ferrets exposed to animals infected with a human H7N9 strain isolated form a patient in Anhui province (AH/1).
To assess reproducibility, they repeated the aerosol transmission test with the AH/1 isolate and got the same result.
Senior author Hualan Chen, PhD, told CIDRAP News that there was no significant difference in transmission among four of the five viruses they tested in aerosol transmission testing. "The transmission of AH/1 to all three ferrets suggests that the H7N9 virus has great pandemic potential," she said.
The team noted that it's difficult to pinpoint which amino acid substitution alone makes the virus highly transmissible, but the amino acid differences between the avian viruses and the Anhui virus range from 1 to 27, suggests that only a few changes are needed to make the virus highly transmissible in mammals.
"Moreover, these changes can occur easily during replication in humans," they added.
Overall, the team said their tests found that the H7N9 viruses from poultry and humans can bind to human airway receptors and can replicate efficiently in ferrets, and that one human isolate can transmit efficiently among ferrets by aerosol droplets.
Chen said she was surprised that all of the viruses tested are able to bind to humanlike receptors and that the PB2 gene of the virus so easily gains mutations during replication in humans that boost its virulence and transmissibility.
Experts weigh pandemic potential
She said the group's findings are useful for weighing the threat from the virus. "This study suggests that the H7N9 virus is likely to transmit in humans, and immediate action, not only in China, is needed to prevent a possible pandemic caused by such a virus," Chen said.
The ability of the virus to transmit easily in poultry across a large part of China over a brief period points to the importance of control measures in poultry markets, the group said. But stamping out H7N9 will be a big, long-term challenge, because the virus spreads silently in chickens and also spreads to humans.
Ian Mackay, PhD, a virologist at the Australian Infectious Diseases Research Centre at the University of Queensland, told CIDRAP News that the study's molecular epidemiology component is the largest of its kind to date and adds many more complete H7N9 genomes to the publicly accessible GenBank database. Mackay also authors the Virology Down Under Web site.
He noted that the group's comparison between the human and avian strains found that they differed by less than 4% at the nucleotide level, "Sometimes there are no differences," he said, noting that the most divergent strains came from the Shanghai region, where 20% of the samples originated.
The team's infection experiments on chickens confirm that H7N9 is a silent spreader and that the birds shed the virus for about a week before their illness resolves, Mackay said.
Findings revealed that major differences between H7N9 viruses are at the amino acid level, with the most divergent segment at the PB1 gene. "But it is the PB2 and HA segment that harbors mutations of particular interest to document the journey from infrequent spillover events to sustained human-to-human pandemic-level transmission," he said.
Mackay added: "We know that pandemic potential does not rest solely on one or other amino acid change, but rather a collection of changes. We also don't know what we don't know yet."
The Harbin group showed the potential of H7N9 viruses to bind to both avian and human receptors and that human isolates replicated well in the upper airways, a site for efficient transmission, he noted.
"The study reinforces that even 'lowly' or inefficient transmission—only 33% of ferrets, for example—is still transmission," Mackay said. "That proportion would lead to a lot of human cases in densely populated or frequented areas."
Those factors might help explain the wide clinical spectrum that has been seen, as well as difficulties in tracking the source and the proportion of patients who get severely ill and die, he said.
Zhang Q, Shi J, Deng G, et al. H7N9 influenza viruses are transmissible in ferrets by respiratory droplet. Science 2013 Jun 18 [Abstract]
Jul 10 CIDRAP News story "New studies on H7N9 raise pandemic concerns"
Virology Down Under Web site