Novel Reassortant Highly Pathogenic Avian Influenza (H5N5) Viruses in Domestic Ducks, China

In China, domestic ducks and wild birds often share the same water, in which influenza viruses replicate preferentially. Isolation of 2 novel reassortant highly pathogenic avian influenza (H5N5) viruses from apparently healthy domestic ducks highlights the role of these ducks as reassortment vessels. Such new subtypes of influenza viruses may pose a pandemic threat.

In China, domestic ducks and wild birds often share the same water, in which infl uenza viruses replicate preferentially. Isolation of 2 novel reassortant highly pathogenic avian infl uenza (H5N5) viruses from apparently healthy domestic ducks highlights the role of these ducks as reassortment vessels. Such new subtypes of infl uenza viruses may pose a pandemic threat.
A quatic birds are considered the natural reservoir for infl uenza A viruses of all known 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes (1). Infl uenza viruses replicate preferentially in the cells lining the intestinal tracts of wild waterfowl, usually causing no clinical signs. Excretion of substantial amounts of virus in the feces can infect wild and domestic birds by waterborne transmission (1). In the People's Republic of China, domestic ducks raised in the traditional free-range system often share water with wild aquatic birds. Moreover, domestic ducks are often in close contact with poultry, livestock, and humans in the same village or farm. Therefore, domestic ducks play a major role in the ecology of infl uenza viruses (2) and can act as potential vessels for genetic reassortment (3). Systematic surveillance of infl uenza viruses in domestic ducks could provide timely and valuable epidemiologic information and should be continued.

The Study
As part of routine surveillance for avian infl uenza viruses from December 2008 through January 2009 in eastern China, tracheal and cloacal swab samples from apparently healthy domestic ducks in live poultry markets were collected for virus isolation and identifi cation as described (4). From these samples, 2 infl uenza (H5N5) These 2 viruses grew effi ciently in eggs and in MDCK cells, each with virus titers >8 log 10 50% egg infectious dose (EID 50 )/mL or 8 log 10 50% tissue culture infectious dose/mL ( Table 1). The intravenous pathogenicity index for chickens and 50% lethal dose for mice were 2.6 and 10 4.0 EID 50 for 008 (H5N5), 2.5 and 10 5.4 EID 50 for 031 (H5N5), respectively (Table 1). Therefore, both novel infl uenza subtype H5N5 viruses were assumed to be highly pathogenic for chickens and moderately virulent for mice (5).
When mice were inoculated with a sublethal dose of 10 3.5 EID 50 , each infl uenza subtype H5N5 virus was able to replicate without prior adaptation. The highest virus titers were detected in the mouse lung. The viruses were able to spread to the brain and heart. Furthermore, infl uenza virus 008 (H5N5) was isolated from the spleen and liver, and infl uenza virus 031 (H5N5) was detected in the kidney (Table 1). Microscopic fi ndings in infected mice were interstitial pneumonia with various amounts of erythrocytes in alveolar lumens, hyperemia and lymphocyte infi ltration of meningeal veins and cardiac muscles, low numbers of lymphocytes in periarterial lymphatic sheaths and macrophage recruitment in the spleen, lymphocyte infi ltration in the liver, and slight congestion in the renal cortex and glomerulus (data not shown).
Genomic analysis showed that the infl uenza viruses 008 (H5N5) and 031 (H5N5) were highly homologous with each other, sharing 99.2%-99.7% nt identities among the 8 gene segments except for the polymerase acidic protein (PA) gene (94.1%). Another  )-were more closely related to the novel infl uenza subtype H5N5 viruses than were those in GenBank ( Table 2).
As outlined by the World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization unifi ed nomenclature system for subtype H5N1 highly pathogenic avian infl uenza (HPAI) viruses (6), the HA genes of the 2 subtype H5N5 viruses were classifi ed into clade 2.3.4 ( Figure 1, panel A), which has been the prevalent lineage in southern China since 2005 (7)(8)(9). In addition to the typical residues Q226 and G228 in HA, which confer receptor preference for SAα2,3Gal, infl uenza viruses 008 (H5N5) and 031 (H5N5) simultaneously carried an S227R mutation in the receptor-binding pocket. A recent report (10) indicated that the S227N substitution accompanied with deglycosylation at residue 158 could substantially increase the affi nity of HA for SAα2,6Gal without reducing its binding affi nity for SAα2,3Gal. Whether this S227R variation with the changed residual polarity affects the receptor-binding property deserves further investigation.
To construct the NA tree, we retrieved 90 complete N5 sequences from GenBank, including the only 2 infl uenza subtype H5N5 viruses from the United States: A/mallard/ MN/105/2000 and A/duck/Massachusetts/sg-00440/2005. The N5 viruses were grouped into 2 lineages-North American and Eurasian-in accordance with their geographic distribution (Figure 1, panel B). The 2 subtype H5N5 isolates from China belonged to the Eurasian lineage, whereas the 2 from the United States clustered within the North American lineage. In addition, 1 aa deletion at residue 42, located in the stalk region of NA, was identifi ed in all Eurasian, but not in the North American, strains.

Conclusions
The 2 novel HPAI (H5N5) viruses isolated and characterized in this study are most likely reassortants of recent Eurasian viruses sharing approximate spatiotemporal distribution. It is less likely that they were introduced through intercontinental transmission of subtype H5N5 strains from North America. Considering the endemicity that clade 2.3.4 subtype H5N1 viruses have gained in China since 2005 (7-9), it is plausible that subtype H5N1 viruses have provided the backbone for generating the novel subtype H5N5 viruses instead of the opposite gene fl ow.
Ducks have been considered "Trojan horses" for infl uenza (H5N1) because of their pivotal role in virus propagation and evolution (11)(12)(13) surveillance should be instituted to identify emerging HPAI (H5N5) viruses and to reduce their potential threat to animal and human health.  All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required.
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