Reassortment of American and Eurasian genes in an influenza A virus isolated from a great black-backed gull (Larus marinus), a species demonstrated to move between these regions
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- Wille, M., Robertson, G.J., Whitney, H. et al. Arch Virol (2011) 156: 107. doi:10.1007/s00705-010-0839-1
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The primary hosts for influenza A viruses are waterfowl, although gulls and shorebirds are also important in global avian influenza dynamics. Avian influenza virus genes are separated phylogenetically into two geographic clades, American and Eurasian, which is caused by the geographic separation of the host species between these two regions. We surveyed a gregarious and cosmopolitan species, the Great Black-backed Gull (Larus marinus), in Newfoundland, Canada, for the presence of avian influenza viruses. We have isolated and determined the complete genome sequence of an H13N2 virus, A/Great Black-backed Gull/Newfoundland/296/2008(H13N2), from one of these birds. Phylogenetic analysis revealed that this virus contained two genes in the American gull clade (PB1, HA), two genes in the American avian clade (PA, NA), and four genes in the Eurasian gull clade (PB2, NP, M, NS). We analyzed bird band recovery information and found the first evidence of trans-Atlantic migration from Newfoundland to Europe (UK, Spain and Portugal) for this species. Thus, great black-backed gulls could be important for movement of avian influenza viruses across the Atlantic Ocean and within North America.
Wild birds are the primary reservoir for influenza A viruses and typically carry low-pathogenic strains (LPAI), which can mutate and produce highly pathogenic strains (HPAI) . LPAI viruses have been isolated from at least 105 wild bird species across 26 different families . The highest prevalence of infection occurs in waterfowl (Anseriformes) worldwide and shorebirds (Charadriiformes) in North America . In addition to birds, influenza A virus infects numerous host species, including humans, pigs, horses, minks, marine mammals, cats, and dogs [1, 3, 4]. Influenza A is a re-emerging disease in humans, causing yearly epidemics and also more irregular pandemics. The continual circulation of influenza A in host populations is maintained through the dynamic evolutionary capacities of the virus, with changes occurring through mutation leading to immune evasion (antigenic drift) and through genome segment reassortment after coinfection by more than one virus (antigenic shift) . These also contribute to the capacity of influenza A to move between host species [1, 6].
Avian influenza A viruses (AIV) are broadly divided into two clades, American and Eurasian. This is due to limited overlap in North American and European bird migration flyways . Exceptions occur in the Beringia region of Alaska and Russia [7–9], and numerous species, particularly waterfowl and shorebirds, move between Asia and Alaska each year . Due to the overlap of old-world and new-world migratory pathways in Alaska, the likelihood of intercontinental reassortment events is hypothesized to be greater in this area [7, 9–11]. Unlike the Beringia area, the North Atlantic Ocean is a very large expanse of water between continents with few opportunities for birds to stop on land. Therefore, only highly adapted pelagic seabirds, including some gull species, which are able to spend large amounts of time on the water inhabit the North Atlantic. The prevalence of avian influenza is low in both gull (<0.1–13%) and seabird (<0.1–4.26%) species [2, 7, 12–15]. However, a recent serological study indicated that large proportions of individuals in gull populations have been infected with AIV at some point in time . With low prevalence of infection, it would seem less likely that reassortment events occur in these bird groups. However, genome sequences of AIV from Delaware Bay on the coastal Atlantic US indicate that intercontinental reassortment is occurring across the Atlantic Ocean [12, 13, 16–19].
The islands along eastern Newfoundland contain the most important concentration of breeding seabirds in eastern North America . This, in addition to open landfill sites and traditionally large fisheries, results in large breeding and wintering gull populations [21–24]. Species such as Great Black-backed Gull (Larus marinus) not only breed on the island of Newfoundland, but also maintain large overwintering populations on the island. Arctic-breeding gull species and small numbers of various Eurasian gull species overwinter on Newfoundland. The timing of the movements of these species is such that these American and Eurasian groups often overlap on the island of Newfoundland during spring and fall months, and therefore it could be an important area for transmission of viruses from one group of gulls to another. All of these species intermingle with waterfowl at onshore water bodies as well, further increasing the opportunity for transmission and reassortment.
We have been conducting surveillance for AIV in wild birds in coastal Newfoundland and Labrador, largely focused on aquatic bird species such as seabirds and gulls that move large distances within the North Atlantic region. Through this work, we have identified two AIV-positive great black-backed gulls amongst 38 of this species tested in Newfoundland between June 2008 and March 2009. We have sequenced the complete genome of one virus that could be cultured and used phylogenetic analysis to show that it contains several Eurasian gull-like segments. We have also analyzed bird band return information for this species, which shows that this species does move across the North Atlantic and therefore has the potential to move viruses between America and Eurasia.
We tested 38 great black-backed gulls in Newfoundland (Canada) for the presence of avian influenza in the 2008–2009 sampling season (i.e., June 2008 through March 2009). We captured and sampled 18 chicks on Gull Island (47°16′N, 52°46′W) during June and July 2008 and collected 8 dead gulls (adult and immature birds) in October of 2008 in St. John’s (47°34′52″N, 52°40′29″W). Another 12 birds were captured at the St. John’s landfill between January and March 2009. The openings of the cloaca and the pharynx of all dead birds were swabbed with a sterile-tipped applicator, which was then inserted into a tube containing Multitrans viral transport media (VTM) (Starplex Scientific, Etobicoke, Canada). Pharyngeal swabs were not collected from live birds. Tubes containing samples were kept cool and placed at −80°C within 24 h of collection.
Sample screening and virus culture
RNA was extracted from an aliquot of VTM from each swab sample using a MagMAX-96 Viral RNA Isolation Kit (Ambion, Streetsville, Canada) with an elution volume of 50 μL. Samples were assayed for the presence of the avian influenza matrix gene by real-time RT-PCR (rRT-PCR)  using a QuantiTect Probe RT-PCR Kit (Qiagen, Mississauga, Canada) in a reaction volume of 20 μL.
Virus isolation was carried out in 9- to 11-day-old SPF embryonated chicken eggs (Charles River, North Franklin, Connecticut) inoculated via the allantoic route. The eggs were then candled daily to monitor for embryo mortality. Two blind passages were performed, and allantoic fluid was tested for hemagglutinating activity after each passage.
RNA isolation and amplification of virus gene segments
Allantoic fluid was mixed with an equal volume of TriPure Isolation Reagent (Roche, Mississauga, Canada), and RNA was extracted using a MagMax AI/ND Viral RNA Isolation Kit (Ambion) following the manufacturer’s instructions. cDNA was synthesized from the RNA sample using the Uni12M primer  and the Superscript III First Strand Synthesis System for Reverse Transcriptase PCR (Invitrogen, Burlington, Canada). Twenty-eight PCR reactions were then carried out in order to amplify the entire genome from this cDNA using a combination of primers (Online Resource 1) [8, 27–33]. PCR products were purified using a QIAquick PCR Purification Kit (Qiagen). Capillary sequencing of PCR products was carried out at The Centre for Applied Genomics (Toronto, Canada). Complete segment sequences were assembled using Geneious v3.8.5 (Biomatters, New Zealand).
The placement of the great black-backed gull virus segment sequences within specific AIV lineages (i.e., American avian, Eurasian avian, American gull or Eurasian gull) was done by phylogenetic analysis of representatives of these groups. Ten American avian and Eurasian avian reference sequences were selected from the NCBI Influenza Virus Resource Database . Representative virus sequences were selected from those isolated from duck species within the last 20 years. Sequences were selected from across North America and included those from Alaska, Alberta, Minnesota and New York. Similarly, the selected Eurasian sequences were isolated in Russia, China and Japan. All available H13 and H16 gull viruses (as of November 2009) were also included (Online Resource 2). Sequences were aligned using ClustalW version 1.4, and the resulting alignments used to construct neighbour-joining trees  with 10,000 bootstrap replicates , all done within MEGA 4.0 . To provide a more detailed view of the relationships between A/Great Black-backed Gull/Newfoundland/296/2008(H13N2) and other viruses, the subclades containing A/Great Black-backed Gull/Newfoundland/296/2008(H13N2) were subsequently reanalyzed using MrBayes v3.1.2 [38, 39] with 2,000,000 generations. Sequences from A/duck/Novosibirsk/02/05(H5N1), falling within the Eurasian avian clade, were used as outgroups for the PB2, PB1, PA, NP, M, and NS segments. The H16 and N3 gene segments from A/shorebird/Delaware/168/06(H16N3) were used as outgroups for the HA and NA trees, respectively.
Bird band returns
All Newfoundland and Labrador great black-backed gull banding (marked with individually coded rings placed on their legs) and encounter (report of banded bird) data were requested from the Bird Banding Office, Canadian Wildlife Service, Environment Canada. This comprised 3012 bird banding records and 69 encounter records from 1928 to July 2009. Bird banding effort and encounters were categorized into the Avalon Peninsula (46°43′N–48°12′N, 52°46′W–54°13′W), central Newfoundland (47°54′N–49°44′N, 54°14′W–57°33′W), Burin Peninsula (46°46′N–47°54′N, 54°27′W–56°01′W), western Newfoundland (47°29′N–49°36′N, 57°34′W–58°27′W), the Northern Peninsula (49°37′N–51°37′N, 57°34W–58°4’W), and southern Labrador (51°29′N–52°53′N, 55°40′W–57°5′W). ArcGIS 9.3  was used to build the map using a polar orthographic projection.
Data for great black-backed gulls banded in Europe and encountered in Greenland, Iceland and North America were requested from EURING, the authority that maintains bird banding information in Europe .
Avian influenza prevalence in great black-backed gulls
In total, 38 samples were collected from great black-backed gulls between June 2008 and March 2009 in Newfoundland and Labrador, Canada. Two individuals sampled in October tested positive for avian influenza; all birds sampled in October (n = 8) had died due to aspergillosis, a fungal infection caused by Aspergillus fumigatus. No viruses were detected in samples taken from chicks during the summer months on island breeding colonies (n = 18) or from birds spending the winter in St. John’s, Newfoundland (n = 12).
One virus was successfully cultured and subtyped as H13N2 by sequencing the HA and NA segments [A/Great Black-backed Gull/Newfoundland/296/2008(H13N2)]. The second positive sample (A/Great Black-backed Gull/Newfoundland/355/2008) was weakly positive in the rRT-PCR assay and presumably contained a very low titer of virus. Attempts to culture virus from this weakly positive sample were unsuccessful, and we were also unable to amplify any gene segments from the swab sample by conventional RT-PCR; therefore, no subtype or sequence information could be obtained.
Relationship of A/Great Black-backed Gull/Newfoundland/296/2008(H13N2) to previously characterized viruses
Neuraminidase subtypes of H13 AIV
Great black-backed gull movement
The AIV isolated from a great black-backed gull in Newfoundland contains four segments of Eurasian viral descent. However, it is overall most closely related to another AIV isolated from coastal Atlantic North America, A/shorebird/Delaware/168/06(H16N3), with five segments of the two viruses (PB2, PB1, NP, M, and NS) showing close relationships. Therefore, these two viruses clearly share a recent common ancestor. However, there has also been reassortment that introduced different PA, HA and NA segments between the viruses. Overall, the patterns we have observed indicate that some of these Eurasian segments have been circulating within North America for some time, and thus the great black-backed gull virus is unlikely to represent a direct arrival from Eurasia.
Despite a high level of AIV surveillance effort in North America and Eurasia, a lack of targeted effort on gull populations, particularly in North America, is evident when analyzing the H13 phylogenetic tree. The virus from Newfoundland is the first found in 20 years in North America containing an H13 gene with a distinctive sequence insertion. It is likely that these genes have indeed been circulating within North American gulls, but just that no viruses containing this unique gene have been detected.
Rather than following classical intracontinental migration patterns, numerous gull species have migration routes that include both intercontinental and intracontinental movements . Although the majority of the great black-backed gulls from Newfoundland move along coastal Atlantic North America on an annual cycle, the bird band return data also clearly demonstrate trans-Atlantic movements in this species as several individual birds that were banded in Newfoundland were subsequently observed in Europe (~5% of the bird band returns). The subset of birds that move across the Atlantic are likely to comprise individuals that breed at the eastern edge of Newfoundland and have only recently been detected due to an increase in bird banding effort in this area . No such intercontinental movement of great black-backed gulls has been recorded from populations in the Maritime provinces of Canada  or the United States . Although great black-backed gulls banded in Europe have not been found in North America, they do move large distances and have been recorded as far west as Greenland.
Our analyses provide evidence that long-distance gull migration is contributing to movement of AIV genes between Eurasia and America. With the evidence that great black-backed gulls are moving across the Atlantic Ocean, they may be playing an important role in moving viruses between these regions, as well as further circulating them within North American gulls and shorebirds during intracontinental movements. It is clear that AIV genome segments that appear to have originated in Eurasian gulls have moved to the North American system and are now circulating within that system. Increased surveillance in gulls for AIV, including in the northeastern United States and Canada, will be an important step towards better understanding the role of gulls in global AIV dynamics. This will also increase our understanding of intercontinental gene exchange and will aid in assessing the risk of invasion of potentially dangerous viruses and/or gene segments.
We thank P. Ryan, A. Granter, C. Keane and A. Blundon for assistance with sample collection and processing, the Environment Canada Banding Office and C. du Feu from EURING for assistance with bird banding and band recovery data, and G. Humphries for assistance with GIS-based data visualization. Support for this work was received from the Newfoundland and Labrador Department of Natural Resources and the Strategic Applications of Genomics in the Environment (STAGE) program at Environment Canada. MW was supported by a CGS-M fellowship from the Natural Sciences and Engineering Research Council (NSERC) and a merit fellowship from Memorial University. Research in ASL’s lab is supported by grants from NSERC, the Canada Foundation for Innovation, and the Industrial Research and Innovation Fund from the Government of Newfoundland and Labrador.