Isolation of a reassortant H13N2 virus from a mallard fecal sample in South Korea
© Kang et al.; licensee BioMed Central Ltd. 2012
Received: 25 January 2012
Accepted: 27 June 2012
Published: 23 July 2012
Virus subtype H13N2, A/mallard/Kr/SH38-45/2010 (H13N2), was first isolated from a mallard fecal sample in South Korea.
Phylogenetic analysis of all eight viral genes revealed that this virus emerged by genetic mixing between Eurasian and North American gene pools, and possibly between wild ducks and gulls. The H13 and N2 surface genes clustered together in a group with Eurasian isolates from gulls and wild birds, respectively. The PB2, PA, NP, M and NS segments belonged to the Eurasian lineage, whereas the PB1 gene clustered in the North American lineage. Furthermore, they showed a bird-dependent pattern in phylogenetic analysis: the M gene was similar to subtype H13 viruses within gulls, whereas other segments were similar to avian influenza viruses of other subtypes from wild ducks.
The data suggests that the novel reassortant H13N2 virus isolated in South Korea might have emerged by genetic reassortment between intercontinental and interspecies transmission in wild birds.
Wild birds in the orders Anseriformes (ducks, geese and swans) and Charadriiformes (gulls, terns and shorebirds) collectively are the natural reservoir for all known subtypes of avian influenza viruses [1, 2]. Influenza virus subtype H13 seems to be highly gull-associated [2, 3] and is rarely found in other avian species that are natural hosts of influenza A virus, such as ducks and geese [4, 5]. Since the first report from the United States in 1982, H13 influenza virus has been detected in Eurasia, and North and South America, with corresponding evolutionary lineages .
In South Korea, fresh fecal samples from migratory wild birds were collected during wintering seasons, from September 2010 to the following March to survey for avian influenza viruses (AIVs) in four wetlands (Miho-cheon, Pungse-cheon, Shihwa-ho and Cheonsu-man). Fresh fecal samples from migratory birds such as mallard (Anas platyrhynchos) and spot-billed duck (Anas poecilorhyncha), visually identified, were collected from the environment. However, as we actually collected samples from a group of birds after observing defecation, we need confirming bird species by mitochondrial analysis. AIVs were isolated by embryonic egg inoculation with fecal samples obtained from migratory wild birds and the presence of AIV was determined using a hemagglutination assay after incubating the eggs at 37 °C for 4–5 days. AIVs were subtyped using reverse transcription-polymerase chain reaction and DNA sequencing. A barcoding system utilizing mitochondrial DNA of bird feces [6, 7] was employed to determine the host species. The viral genes were sequenced and analyzed as previously described . The gene sequences of this H13 virus have been deposited in GenBank under accession no. (JX030402 ~ JX030409).
Within this collection, a total of 2,227 fecal samples were collected and AIV was recovered from 59 samples. H13N2 virus [A/mallard/Kr/SH38-45/2010 (H13N2), designated Md/SH38-45 (H13N2)] was identified. Although intensive surveillance on wild birds for avian influenza has been performed, there has been no prior report of the isolation of H13 virus in South Korea [9–11]. Subtypes H13 and H16 may be highly associated with gulls . Therefore, the failure to detect subtype H13 may be associated with the AIV surveillance program in South Korea, which has largely focused on fecal samples of migratory wild ducks. To our knowledge, this is the first report of the isolation of a H13 subtype virus in South Korea.
Lineage analysis of Md/SH38-45 (H13N2) virus
The present analyses revealed that internal genes, polymerase subunit PB2 (PB2), polymerase acidic (PA), nucleoprotein (NP) and nonstructural protein (NS) genes belonged to the Eurasian lineage, and that they were similar to AIVs from wild ducks ( Additional file 1: Figures S1b, d, e, g). The M segment clustered in a group with the Eurasian lineage and was similar to subtype H13 viruses within gulls ( Additional file 1: Figure S1f). In contrast, the PB1 gene clustered in the North American lineage, unlike the other genes, and showed high sequence similarity with the isolates from wild ducks ( Additional file 1: Figure S1c). In addition, two viruses [glaucous gull/Alaska/44199-006/06 (H13N9) and glaucous gull/Alaska/44199-104/06 (H13N9)] from Alaska  that were included in the phylogenetic analyses were closely related with four genes [HA (97.1–97.2%), NP (98.1–98.3%), M (96.6–96.7%), and NS (98.0–98.1%) of Md/SH38-45 (H13N2) virus, showing high sequence similarity.
Together, our findings indicate that the overlap of the East Asian/Australian and the Pacific Americas migratory flyways may serve as not only a link between continental AIV gene pools in waterfowl hosts, but also in gulls [7, 13]. The PA segment of internal genes belonged to the same lineage (94.8–96.6% homology) with the Gs/GD-like lineage of H5N1 highly pathogenic avian influenza viruses ( Additional file 1: Figure S1d). This result is consistent with an earlier report that the multiple gene pools of wild birds provide Gs/GD-like viruses with their genes in part by reassortment events .
In conclusion, it seems likely that reassortment events of Md/SH38-45 (H13N2) virus have emerged as the result of genetic mixing between Eurasian and North American gene pools, and between wild ducks and gulls.
This work was supported by a grant from Animal, Plant and Fisheries Quarantine Inspection Agency (C-AD15-2010-12-01), Republic of Korea.
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