Study finds H1N1 and H9N2 offspring can be more lethal

Editor's note: This story was revised Mar 3 to include comments on the study from a virologist who was not involved in it.

Mar 2, 2011 (CIDRAP News) – Chinese researchers who tested reassortants that combined the 2009 H1N1 virus with H9N2, a subtype that commonly circulates in birds, found that several were more pathogenic then the parent viruses, which they said could pose a pandemic threat.

Their experiments also found that of 127 reassortants between the two viruses, as many as 57.5% had a high ability to replicate, similar to the two parent viruses, which suggests that the two viruses have a high genetic compatibility. The findings appeared in the Proceedings of the National Academy of Sciences. The report was edited by Dr Peter Palese, a well-known virologist at Mount Sinai School of Medicine in New York City.

Though H9N2 is a low-pathogenic virus, it has also been found in pigs, which infectious disease experts have said could be a "mixing vessel" for animal and human flu viruses, producing novel flu viruses with pandemic potential.

Human infections with H9N2 have been reported, and the World Health Organization (WHO) includes the virus alongside H5N1 in its annual recommendations for pandemic flu vaccine candidate viruses.

Unpacking virulence differences
Using reverse genetics, the study group attempted to make all possible 127 reassortants from an avian H9N2 influenza virus and the 2009 H1N1 virus. Then they categorized each into one of four groups based on replication ability.

A total of 73 reassortants showed a high ability to replicate, and the group evaluated the pathogenicity of all of them in mice. Based on those findings, they sorted the viruses into three groups that varied by how sick the mice got. Eight of the reassortants caused more severe disease, based on clinical observation such as ruffled coat, lethargy, and dyspnea, and microscopic signs in lung tissue, which included interstitial pneumonia and bronchopneumonia, edema, hemorrhage, epithelial cell dropout, and infiltration of inflammatory cells.

All eight of the most pathogenic reassortants had the polymerase acid (PA) gene segment from the 2009 H1N1 virus, which the authors suggest is required for the emergence of the most virulent reassortants they tested. Further analysis showed that the basic polymerase 1 (PB1) gene of the 2009 H1N1 virus usually attenuated the pathogenicity of the reassortants, and the neuraminidase of that parent virus typically increased their virulence.

Projecting illness impacts
Though the more virulent reassortants were a major concern, the less pathogenic ones that emerged are also worth noting, the authors state, because they could circulate undetected in mammals, and mutation or further reassortment could also pose a pandemic threat.

Not all of the reassortants that had the PA gene segment from the 2009 H1N1 virus were more virulent that their parent viruses, which the investigators said suggests that other gene segments play a role in pathogenicity.

They said their examination of the sickest mice showed no evidence of systemic spread, but all viruses replicated efficiently in the lungs and showed high polymerase activity. "These results suggested that a high virus load and high polymerase activity were important factors for the virulence of the reassortants in mice," they wrote.

The group concluded that reassortants between H9N2 and pandemic H1N1 could pose a public health threat and that the PA gene segment findings in the most virulent ones could serve as a marker for identifying H9 reassortants that present the greatest risks.

Expert says study breaks new ground
Vincent Racaniello, PhD, professor of microbiology at Columbia University and author of Virology Blog, told CIDRAP News that the study was "heroic," given that the authors made all possible combinations of RNA segments among H1N1 and H9N2. He said the high compatibility between the two strains was unexpected. "But I'm not aware of any other study in which all possible 127 reassortants were generated. Perhaps similar results would be found for other combinations," he said.

It's not clear if the higher pathogenicity of the eight ressortants compared with the parent viruses was unique to 2009 H1N1 and H9N2 or if investigators would find similar results by testing other parent pairs, Racaniello said.

He said he wasn't worried about the pandemic potential of such reassortants for two reasons: findings in mice don't necessarily apply to pathogenesis in humans, and the most important property for the selection of new flu virus variants is transmission, not virulence. "Being virulent doesn't help a virus if it can't transmit," he said.

The finding that the PA gene from the H1N1 virus was required for increased virulence was interesting, Racaniello said. "In 2009 H1N1 the PA gene is of avian origin, and may be more compatible with other avian genes. The avian polymerase is restricted in human cells, and this restriction can be overcome by the presence of the human PA subunit."

The 2009 H1N1 PA gene's effect may be to produce a more efficient polymerase that allows higher viral replication and enhances virulence, though the effect on transmission isn't known, he said. "I think this result would have implications only in cases of reassortants with avian viruses, or viruses with an avian polymerase."

New study plots out H9N2 family tree
In a related development, researchers said that the extensive reassortments that the H9N2 flu virus has undergone shows it has the potential for continued influenza outbreaks in poultry and epidemics in humans.

A group from China that included two scientists from the US Department of Agriculture conducted a sequence analysis of 571 viral genomes from a flu virus database to pinpoint the subtype's genetic and evolutionary characteristics.

They found 74 separate lineages, which showed host and geographic differences. Genotype analysis revealed at least 98 different ones, which fell into seven series according to their hemagglutinin lineages.

Analyses of the internal genes found that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtypes.

Because H9N2 viruses have undergone extensive changes that produced many reassortants and genotypes, continued circulation in diverse hosts could lead to future outbreaks in poultry and humans.

They wrote that their analyses offers the world's first full characterization of H9N2's phylogeny and genetic diversity, and they proposed a nomenclature system for identifying all of the subtype's lineages and genotypes that could be helpful for epidemiology, evolution, and ecology studies.

New information from the study suggests that the geographic distribution of H9N2 viruses is larger than earlier studies reported, spanning Asia, the Middle East, Europe, Africa, and North America, the authors wrote. Some North American H9N2 viruses have links to Asia and Europe, which they said suggests that the H9N2 viruses have become more complicated.

The phylogenic analysis suggests that the H9N2 virus is very diverse, with permanent lineages established in Asian poultry and with frequent appearances in pigs, especially in China.

Sun Y, Qin K, Wang J, et al. High genetic compatibility and increased pathogenicity of reassortants derived from avian H9N2 and pandemic H1N1/2009 influenza viruses. Proc Natl Acad Sci 2011 (published online Feb 28) [Abstract]

Dong G, Luo J, Zhang H, et al. Phylogenetic diversity and genotypical complexity of H9N2 influenza A virus revealed by genomic sequence analysis. PLoS One 2011 Feb 28;6(2):[Full text]

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