New studies on H7N9 raise pandemic concerns

Two research teams that conducted a massive number of experiments on the new H7N9 influenza virus found more signs that it could be a pandemic virus, though their animal tests showed that its ability to spread through coughs and sneezes isn't as robust as seasonal flu.

The two studies are among several recent efforts to assess the threat from the new virus, which infected 134 people, 43 of them fatally, before tapering off in early June. Experts aren't sure if the virus has died out or if it has temporarily retreated due to warmer weather and perhaps the effects of outbreak response measures aimed at live-poultry markets in some of China's biggest cities.

Both studies were published in the same issue of Nature. One of the teams is from Japan and the University of Wisconsin, Madison, lab of Yoshihiro Kawaoka, DVM, PhD. That team conducted several types of tests and comparisons on two novel H7N9 strains from China, an earlier avian H7N9 strain, and the 2009 H1N1 virus. They also examined how the new virus behaves in mice, ferrets, miniature pigs, macaques.

The second group includes scientists from the US Centers for Disease Control and Prevention (CDC), which also studied two novel H7N9 strains from China, focusing on how it infects human cells and how it spreads in ferrets and mice.

Pandemic potential in first study

When Kawaoka's team compared two novel H7N9 isolates from the first human cases in Anhui province and Shanghai with an avian H7N9 strain from China and the 2009 H1N1 virus, they found that the new viruses were more pathogenic in mice. All three of the H7N9 viruses that they tested replicated well in the nasal passages of ferrets.

In nonhuman primates, the Anhui and avian H7N9 strains replicated efficiently in both the upper and lower respiratory tracts, unlike human flu viruses, which are only known to infect primates' upper airways. However, when researchers inoculated the nasal passages of miniature pigs, they found that the Anhui strain didn't replicate well.

In their transmission tests the group found that the Anhui strain transmitted in respiratory droplets in one of three ferret pairs. Tests found, however, that the two novel viruses, plus another from Hangzhou province, bind to human receptors, a factor the researchers said may be critical for the viruses to transmit in ferrets.

The team wrote that since the H5N1 virus requires several mutations for aerosol transmission in ferrets, "the pandemic potential of A (H7N9) viruses may be greater than that of the highly pathogenic avian H5N1 viruses."

The investigators also included experiments to test sensitivity to neuraminidase inhibitors and to see if humans have any pre-exisiting immunity or cross-protection against the new virus. They found that the Anhui strain was less sensitive to the drugs than the 2009 H1N1 virus, though both were sensitive to favipiravir, an experimental polymerase inhibitor originally developed by a Japanese pharmaceutical company.

To gauge human immunity to the H7N9 virus, the scientists tested 500 blood samples from various age-groups in Japan collected from 2010 through 2012. They found no antibodies to the Anhui H7N9 strain.

Taken together, the findings suggest that the novel H7N9 strains have several traits in common with human influenza viruses, including efficient binding to human-type receptors, efficient replication in mammal cells, and respiratory droplet transmission in ferrets. The low efficacy of neuraminidase inhibitors and the lack of protection make the new virus "a formidable threat to public health," the researchers concluded.

CDC team finds rapid viral reproduction

The second study was part of an effort by the CDC to better understand the transmissibility and characteristics of the new virus. In a press release today, the CDC said that uncovering clues about the threat in the lab is a critical component of the public health response to the emerging disease threat. The team also conducted several experiments on the Anhui and Shanghai H7N9 strains.

In tests on human airway epithelial cells and ferret respiratory tracts, they found that both H7N9 viruses replicated at higher titers compared with the seasonal H3N2 virus. The H7N9 virus showed a 20- to 400-fold increase in replication after 2 days compared with seasonal flu viruses and other avian strains related to H7N9. When matched against seasonal H3N2, the novel virus showed an 80,000-fold increase in replication at 24 hours, according to the study.

Overall, they found that the H7N9 viruses are able to reproduce quickly and produce large amounts of virus in mammal and human airway cells, but at a higher temperature most consistent with lower airways.

Because conjunctivitis in humans has been reported with other H7 viruses, the team inoculated the eyes of mice and ferrets. Though they didn't find consistent virus replication in ocular tissue in mice, they did detect nasal H7N9 virus titers in the animals 3 to 6 days after eye inoculation. They said the finding suggests that although H7N9 may not maintain the ocular tropism of other H7 viruses, it is capable of using the eye as an entry for respiratory infections.

The CDC said in its press release that the finding supports its recommendation to avoid touching the eyes, nose, or mouth to prevent the spread of infections, as well as its advice for health providers to wear eye protection when caring for confirmed or suspected H7N9 infections.

Tests to see how pathogenic the H7N9 viruses were in ferrets found that unlike the H5N1 virus it didn't spread systemically to the spleen, kidney, liver, or intestinal tract, a signal that it did not cause severe illness. However, the researchers found that the virus was easily able to infect mice and caused a more lethal infection.

Their transmission experiments in ferrets showed the virus spread easily through direct contact, but did not transmit readily through respiratory droplets, which they said was accompanied by low binding specificity for human-type receptors. The ratio of transmission in ferrets in the CDC study—with two of six ferrets showing infection by respiratory droplet—echoed the findings in the Kawaoka study.

The team concluded that their transmission studies are consistent with the lack of sustained human-to-human transmission in China's H7N9 outbreak and that their findings suggest that more adaptation in mammals would need to occur before the virus became as transmissible through respiratory droplets as pandemic or seasonal flu viruses.

Experts weigh in on risk conclusions

Two infectious disease experts who were asked to comment on the Kawaoka study praised the comprehensive series of experiments. Marion Koopmans, DVM, PhD, with the National Institute of Public Health and the Environment in the Netherlands, told CIDRAP News that although some of the study's findings have been described, "the combination is kind of an 'everything you ever wanted to know about H7N9' study."

She said the results show that the circulating novel virus strains can infect mammals, cause significant pathology, and may transmit among mammals, though not as efficiently and only in small number.

Though Koopmans said she found the group's conclusion that the viruses pose a formidable pandemic threat a bit strong, given that animal models remain a challenge and human-to-human transmissibility has been limited, she added: "But this is as far as you can get with animal studies, I think."

Michael T. Osterholm, PhD, MPH, director of the University of Minnesota's Center for Infectious Disease Research and Policy, which publishes CIDRAP News, said that, despite the exhaustive look at the new virus, unanswered questions still remain about what it would take for H7N9 to go from a virus that doesn't readily spread human-to-human to one that does.

The study findings, taken as a whole, support that the virus could move toward efficient human-to-human transmission. "The virus is still in its early days," Osterholm said.

However, he said the group's findings, such as the replication in nonhuman primates, are sobering. "Surely, you have to take the pandemic potential with a great degree of seriousness." He said. "With the data presented here, there's no reason to rule that out."

Like meteorologists who comb through their readings and data to issue warnings about tornadoes, the researchers are saying that the conditions are ripe for the H7N9 virus to become a bigger threat, Osterholm said. "You're seeing all the conditions."

Watanabe T, Kiso M, Fukuyama S, et al. Characterization of H7N9 influenza A viruses isolated from humans, letter. Nature 2013 Jul 10 [Abstract]

Belser JA, Gustin KM, Pearce MB, et al. Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice, letter. Nature 2013 Jul 10 [Abstract]

See also:

Jul 10 University of Wisconsin-Madison news story on the Kawaoka study

Jul 10 CDC press release

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