H7N9 gene tree study yields new clues on mixing, timing

May 2, 2013 (CIDRAP News) – The most detailed genetic analysis so far of H7N9 viruses that have infected humans in China shed more light on how the virus may have emerged—a complex set of events that could have involved mixing between wild and domestic ducks and reassortment in poultry.

In analyzing the family tree of H7N9 and related viruses Chinese researchers also found a long branch between the new virus and avian influenza viruses from wild birds, suggesting that an intermediate host might exist. The study appeared yesterday in an early online edition of The Lancet.

The group used H7N9 genetic sequences from the GISAID database that are linked to the first four human isolates. Using sophisticated analysis tools, they compared the H7N9 sequences with public ones from the Influenza Virus Resource, a flu sequence data source and tool. They used leading-edge techniques to estimate the phylogenies, divergence times, and other evolutionary information for all eight gene segments.

The researchers pointed out that their analysis lacks extensive surveillance data, partly because H7 virus subtypes have low pathogenicity for avian hosts, which can harbor the virus for long periods without showing symptoms. However, the team wrote that the study includes most publicly available related sequences.

The new virus fell within the Eurasian H7 lineage and was most closely related to sequences from ducks in Zhejiang province in 2011, and the team estimated that the most recent common ancestor of the H7N9 virus was in January 2012.

Similarly, their analysis of the N9 phylogenetic tree found the H7N9 virus belongs to the Eurasian lineage. It was closely related to H7N9 viruses from wild ducks in South Korea in February and April of 2011. At that time, the wild ducks were colonized for breeding in southeast China during their northward migration along the East Asian flyway.

The six internal genes of the H7N9 virus clustered with H9N2 viruses from China from at least two different origins: one for the NS gene and one for the rest of the internal genes. The most closely related ones were all from one strain, a virus collected from a brambling in Beijing in November 2012. The group's phylogenetic analysis also found that the H7N9 polymerase gene fell within a clade of chicken H9N2 viruses from near Shanghai in 2012 and Zhejiang province in 2011.

The new virus could have at least four possible genetic origins, which is more than an early analysis of the virus proposed, the group wrote.

The NA gene of H7N9 might have traveled from Europe through bird migration and could have been carried by wild birds. The team said a likely scenario is that wild ducks transferred the viruses to domestic ducks, which share similar behaviors and habitats in eastern China. The ducks could have obtained the HA and NA genes from migratory birds sequentially and simultaneously, such as when large colonies of mixed wild ducks winter in southeast China.

The group noted that a long branch between the new H7N9 virus and those from wild birds raises the possibility of an intermediate host, which touches on a question that flu experts are still puzzling over. Though investigations of human cases have found a link to market poultry and environments, little is known about how the market birds are becoming infected.

Taken together, the group wrote that the evidence so far suggests that new H7N9 virus emerged from an HA gene that was circulating in the East Asian flyway in wild birds and ducks, from an NA gene that was introduced from a European lineage early and transferred from migrating wild birds, and from H9N2 viruses in eastern China's chickens and ducks that possibly reassorted with H7 and N9 viruses in ducks.

"After these reassortment events, the new viruses started to circulate in chickens, with low pathogenicity," they wrote, adding that the reassortment events probably occurred in Shanghai and nearby provinces.

The time to the most recent common ancestor for the gene segments, as well as frequent poultry transportation, might have played a role in the increasing sporadic detections of the H7N9 virus in humans, according to the report.

The diversity that they found in the isolates suggests H7N9 has evolved into at least two lineages, with the possibility of unknown intermediate hosts, pointing to a need for heightened global surveillance in animals and humans, they wrote.

Liu D, Shi W, Shi Y, et al. Origin and diversity of novel avian influenza A H7N9 viruses and causing human infection: phylogenetic, structural, and coalescent analyses. Lancet 2013 May 1 [Abstract]

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