Study yields highly pathogenic avian, human flu virus mix

Feb 22, 2010 (CIDRAP News) – Human seasonal flu strains that reassort with avian H5 influenza can produce a more pathogenic avian flu strain, highlighting the importance of virus genetic surveillance and the need to protect people who have close contact with birds, researchers reported today.

Using reverse genetics, researchers generated 254 combinations of reassortant viruses between a low-pathogenic avian H5N1 virus and a human seasonal H3N2 virus. Then they tested the pathogenicity in mice. The study, by a group from Dr Yoshihiro Kawaoka's lab at the University of Wisconsin, Madison, appears in an early online edition of Proceeding of the National Academy of Sciences (PNAS).

Researchers did not conduct tests with the pandemic H1N1 virus, but Kawaoka said in a press release that he worried that, while H5N1 virus has never acquired the ability to transmit among humans, reassortment with the pandemic H1N1 virus might give it that ability. Kawaoka is a professor of pathobiological sciences at the UW-Madison School of Veterinary Medicine.

Previous studies of hybrid avian-human influenza viruses generated in the lab have shown that the reassortants were not more virulent than the parent strains.

"With the new pandemic H1N1 virus, people sort of forgot about H5N1 avian influenza. But the reality is that H5N1 avian virus is still out there," Kawaoka said in the press release. "Our data suggest that it is possible there may be reassortment between H5 and pandemic H1N1 that can create a more pathogenic H5N1 virus."

The investigators generated all 254 combinations of reassortants between the low-pathogenic H5N1 strain (A/chicken/South Kalimantan/UT6028/06) and the human seasonal H3N2 strain they selected (A/Tokyo/Ut-Sk-1/07). They produced the virus stocks by growing the viruses in Madin-Darby canine kidney cells. The viruses fell into four groups: highly replicative (96), moderately replicative (68), low replicative (20), and nonviable (70).

They performed minigenome assays to assess if ribonucleoprotein (RNP) activity played a role in the viability and replicative ability of reassortant viruses and found that RNP activity was high in all 96 viruses in the highly replicative group and low in the nonviable group. Kawaoka's group wrote that the findings confirmed their previous findings.

Before they tested the pathogenicity of the reassortant viruses in mice, they assessed the virulence of the two parent viruses. Neither was highly pathogenic in the mice.

For the main portion of the pathogenicity study in mice, they tested 75 hybrids that met their criteria. Forty-five were less pathogenic than the avian H5N1 parent, 8 were as pathogenic, and 22 were more pathogenic.

The researchers found that the common feature of the more pathogenic viruses was that their PB2 gene (polymerase basic protein 2) was from the human seasonal H3N2 parent. Although introducing the H3N2 PB2 gene into the avian H5N1 virus backbone didn't necessarily result in a more pathogenic virus, they said their findings suggest that human PB2 is required for the emergence of substantially virulent hybrids.

"Taken together, our data suggest that human PB2 is indispensible for reassortant viruses to be virulent to mice," they wrote. "Human PB1 alone lacks the ability to increase virus pathogenicity in mice but could do so in cooperation with human PB2."

They added that the findings provide valuable information to help health officials prepare for future pandemics that could be caused by avian H5N1 and seasonal flu strains, including the pandemic H1N1 virus.

The avian H5N1 virus was used in the study, because most recent human H5N1 infections have links to sick poultry or contaminated poultry products, the group wrote. Reassortance of an H5 virus with pandemic potential could occur when a person infected with seasonal flu had contact with H5N1-infected poultry or poultry products.

Introducing the human PB2 segment into the hybrid seems to allow it to grow at lower temperatures of the human upper-respiratory tract, a key to efficient transmission of pandemic viruses in humans, the group wrote.

They concluded that high pathogenicity is an important property that determines the magnitude of a pandemic, and that one could occur from reassortment between avian H5N1 and human H3N2 influenza viruses. The two factors highlight the important of vaccination programs against the two viruses, at least for those such as poultry or healthcare workers who are exposed to avian H5N1 viruses.

Vincent Racaniello, PhD, professor of microbiology at Columbia University and author of Virology Blog, cautioned that animal models don't always predict what will happen in humans. For example, a few months into the H1N1 pandemic Kawaoka's group reported that trials in four animals models—mice, ferrets, macaques, and pigs—suggested a more severe disease than the mild-to-moderate infections healthcare workers were seeing in humans.

Though the relationship between more virulent virus reassortants and the presence of the PB2 gene is not a new finding, Racaniello said Kawaoka's group confirmed the conclusion with a more extensive panel.

It's not clear if researchers would obtain the same results with an H1N1 panel, and scientists are as uncertain now as they were a year ago about whether the pandemic H1N1 virus could create a more pathogenic H5N1 virus, he said.

"In my view, viruses evolve to be maximally transmissible. Becoming increasingly virulent might help a virus become more transmissible, or it might be a by-product," Racaniello said. "But we should not assume that viruses need to cause more severe disease to be successful in humans. They need to replicate and transmit efficiently."

Li C, Hatta M, Nidom CA, et al. Reassortment between avian H5N1 and human H3N2 influenza viruses creates hybrid viruses with substantial virulence. Proc Natl Acad Sci 2010 Feb 22; early online edition [Abstract]

See also:

Jul 13, 2009, CIDRAP News story "Another study suggests greater novel H1N1 pathogenicity"

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