Genesis and spread of multiple reassortants during the
2016/2017 H5 avian influenza epidemic in Eurasia
Samantha J. Lycetta, Anne Pohlmannb, Christoph Staubachc, Valentina Caliendod, Mark Woolhousee, Martin Beerb, Thijs Kuikend,1, and Global Consortium for H5N8 and Related Influenza Viruses
aThe Roslin Institute, University of Edinburgh, EH25 9RG Edinburgh, United Kingdom;bInstitute of Diagnostic Virology, Friedrich Loeffler Institut, D-17493
Greifswald-Insel Riems, Germany;cInstitute of Epidemiology, Friedrich Loeffler Institut, D-17493 Greifswald-Insel Riems, Germany;dDepartment of
Viroscience, Erasmus University Medical Center, 3015 NC Rotterdam, the Netherlands; andeUsher Institute, University of Edinburgh, EH9 3FL Edinburgh,
United Kingdom
Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved July 1, 2020 (received for review February 6, 2020)
Highly pathogenic avian influenza (HPAI) viruses of the H5 A/goose/ Guangdong/1/96 lineage can cause severe disease in poultry and wild birds, and occasionally in humans. In recent years, H5 HPAI viruses of this lineage infecting poultry in Asia have spilled over into wild birds and spread via bird migration to countries in Europe, Africa, and North America. In 2016/2017, this spillover resulted in the largest HPAI epidemic on record in Europe and was associated with an unusually high frequency of reassortments between H5 HPAI viruses and cocirculating low-pathogenic avian influenza viruses. Here, we show that the seven main H5 reassortant viruses had var-ious combinations of gene segments 1, 2, 3, 5, and 6. Using detailed time-resolved phylogenetic analysis, most of these gene segments likely originated from wild birds and at dates and locations that corresponded to their hosts’ migratory cycles. However, some gene segments in two reassortant viruses likely originated from domestic anseriforms, either in spring 2016 in east China or in autumn 2016 in central Europe. Our results demonstrate that, in addition to domestic anseriforms in Asia, both migratory wild birds and domestic anseri-forms in Europe are relevant sources of gene segments for recent reassortant H5 HPAI viruses. The ease with which these H5 HPAI viruses reassort, in combination with repeated spillovers of H5 HPAI viruses into wild birds, increases the risk of emergence of a reassor-tant virus that persists in wild bird populations yet remains highly pathogenic for poultry.
highly pathogenic avian influenza
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emerging infectious diseases|
phylogenetic analysis
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poultry|
wild birdsI
nfection with highly pathogenic avian influenza (HPAI) virus of the H5 A/goose/Guangdong/1/96 (Gs/Gd) lineage can cause severe disease in birds and in mammals, including people (1, 2). Since the beginning of the century, H5 HPAI viruses have re-peatedly spilled over from poultry to free-living wild birds, es-pecially in Asia. This spillover in regions with a high poultry density and intensive interaction between wild bird and poultry populations has altered the epidemiology of H5 HPAI viruses in several ways. First, migration of HPAI virus-infected wild birds acts as a new route of long-distance spread of H5 HPAI viruses into countries with bird populations that were free of these viruses (3). Second, direct or indirect contact with infected wild birds is a new route of HPAI virus incursion into poultry farms (4–7). Third, H5 HPAI virus infection is a new source of con-siderable mortality in wild birds themselves, and it may sub-stantially affect population dynamics of wild birds and threaten highly protected species like the white-tailed eagle (Haliaeetus albicilla) and the peregrine falcon (Falco peregrinus) (8, 9). Be-cause we have a poor understanding of wild birds as a new niche for H5 HPAI viruses, it is difficult to design efficient surveillance programs, as well as effective prevention and control measures. Since 1996, the Gs/Gd lineage of H5 HPAI viruses has evolved rapidly and is now highly diverse. Following the emergence of Gs/Gd, the lineage has evolved into numerous genetically dis-tinct clades (10, 11). Several of these clades have spread via wildbirds from Asia to Europe since 2004: clade 1 in 2004, clades 2.2 and 2.2.1 from 2005 to 2007, clade 2.3.2 from 2008 to 2010, and clade 2.3.4.4 from 2014 to 2019 (1, 3, 12, 13). Gs/Gd lineage H5 HPAI viruses are now endemic in areas of Asia and continue to evolve, so new epidemics are likely to occur.
The routes by which H5 HPAI viruses are carried over long distances, as well as the particular migratory species involved, are still only roughly known. From wintering grounds of migratory birds in Southeast Asia, H5 HPAI viruses are carried north to breeding grounds on the northern parts of the Eurasian and North American continents and then west to wintering grounds in Europe, east to wintering grounds in North America, or back south to wintering grounds in Asia (3). The species involved are thought to be long-distance migrants of the family Anatidae (ducks, geese, and swans). Several species in this family (e.g., Eurasian wigeon [Mareca penelope], Eurasian teal [Anas crecca], and northern pintail [Anas acuta]) have migratory routes that correspond to the observed pattern of virus spread (3), have been found infected with H5 HPAI virus at different locations
Significance
In 2016/2017, highly pathogenic avian influenza (HPAI) virus of the subtype H5 spilled over into wild birds and caused the largest known HPAI epidemic in Europe, affecting poultry and wild birds. During its spread, the virus frequently exchanged genetic material (reassortment) with cocirculating low-pathogenic avian influenza viruses. To determine where and when these reassortments oc-curred, we analyzed Eurasian avian influenza viruses and identi-fied a large set of H5 HPAI reassortants. We found that new genetic material likely came from wild birds across their migratory range and from domestic ducks not only in China, but also in central Europe. This knowledge is important to understand how the virus could adapt to wild birds and become established in wild bird populations.
Author contributions: S.J.L., A.P., M.W., M.B., T.K., and G.C.f.H.a.R.I.V. designed research; S.J.L., A.P., C.S., V.C., T.K., and G.C.f.H.a.R.I.V. performed research; G.C.f.H.a.R.I.V. contrib-uted new reagents/analytic tools; S.J.L., A.P., C.S., V.C., M.W., M.B., T.K., and G.C.f.H.a.R.I.V. analyzed data; and S.J.L., A.P., C.S., V.C., M.W., M.B., T.K., and G.C.f.H.a.R.I.V. wrote the paper.
The authors declare no competing interest.
A complete list of the Global Consortium for H5N8 and Related Influenza Viruses can be found inSI Appendix.
This article is a PNAS Direct Submission.
This open access article is distributed underCreative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Data deposition: Sequences are shared via the EpiFlu database from the Global Initiative on Sharing All Influenza Data and the International Nucleotide Sequence Database Collaboration.
1To whom correspondence may be addressed. Email: t.kuiken@erasmusmc.nl.
This article contains supporting information online athttps://www.pnas.org/lookup/suppl/ doi:10.1073/pnas.2001813117/-/DCSupplemental.
along their respective migratory routes (3), and can be infected with and excrete H5 HPAI virus—at least under laboratory conditions—without showing detectable clinical signs (1, 14–17). In 2016 and 2017, H5 HPAI viruses (belonging to cluster B, Gochang like) spread again widely across Eurasia, causing the largest and most widespread HPAI epidemic ever recorded in Europe: between October 2016 and August 2017, 1,207 indi-vidual HPAI virus outbreaks in poultry holdings were reported in 24 European Union countries, and 1,590 wild bird mortality events were recorded in 29 countries of the European Union and in Switzerland (18). In contrast, relatively few wild birds and poultry farms were affected in 2014/2015: between November 2014 and February 2015, H5N8 HPAI was detected in only 11 poultry farms (turkey, duck, chicken) and other holdings and in a small number of wild waterfowl across five European countries, as well as in two mute swans (Cygnus olor) in Sweden (3, 19), indicating a marked difference between epidemics.
During the 2016 to 2017 epidemic, five reassortant viruses were detected in Germany alone, to which the H5 HPAI virus contributed at least the clade 2.3.4.4 hemagglutinin (HA) gene segment and often, also that clade’s corresponding matrix (M) and nonstructural (NS) gene segments, while cocirculating low-pathogenic avian influenza (LPAI) viruses contributed gene segments coding for the other viral proteins (20). The genetic diversity generated through reassortment plays an important role in the evolution of influenza viruses (21) and may provide the opportunity for the adaptation of H5 HPAI virus to wild bird populations. Despite this, we know little about the genesis and spread of these reassortants. We therefore performed extensive phylogeographic and epidemiological analyses of the 2016 to 2017 H5 HPAI virus epidemic, including analysis of the temporal and geographical spread of the reassortants and their individual gene segments, and of the avian species involved. Our main goals were to estimate where and when these reassortments occurred and to identify which avian influenza viruses from which host species provided gene segments for reassortment. We used ge-netic sequences of avian influenza viruses obtained from poultry and wild birds worldwide and shared through public databases and epidemiological data obtained from the World Organization for Animal Health. We focused on HPAI viruses of H5 clade 2.3.4.4 collected between May 2016 and July 2017. Data were discussed within the Global Consortium of H5N8 and Related Influenza Viruses (3). The aim of this consortium is to foster data exchange and global analysis of H5Nx avian influenza epidemics (3).
Results
Classification of Whole-Genome Sequences of HPAI Virus H5Nx into Reassortants per Gene Segment.Sequence data for individual gene segments 1 (polymerase basic 2, PB2), 2 (polymerase basic 1, PB1), 3 (polymerase acidic, PA), 5 (nucleoprotein, NP), 7 (M), and 8 (NS) were classified into groups based on phylogenetic similarity, and sequence data for gene segment 6 (neuraminidase, NA) were classified by subtype (Materials and Methods). Using BEAST (22), time-scaled trees were inferred for each segment or subtype for segment 6 (Figs. 1 and 2). The discontinuous character of the phylogenetic groups was consistent with the importation of novel sequences (i.e., reassortment or genetic shift), suggesting that these groups corresponded to reassortants (Table 1). These results suggested that segment 4 (the HA gene) had four main combinations (A to D) with segment 1, three (A, C, D) with segment 2, four (A, B, D, E) with segment 3, five (A, B, D, E, F) with segment 5, two (N8 and N5) with segment 6, and one (A) with segments 7 and 8. The phylogenetic analysis of the eight gene segments indicated that, in some cases, multiple gene segments transferred in the same reassortment event. An example is the simultaneous reassortment of gene segments 1, 3, and 5 (Figs. 1 and 2 and Table 1).
By Influenza Reassortment analysis via Supernetworks (IRIS), a total of 446 full-genome sequences of clade 2.3.4.4 H5 HPAI viruses could be divided into 11 distinct reassortants, 7 of which were common. Supernetworks are calculated from all eight segment-sorted maximum-likelihood trees and visualize the phylogenetic relationships of each HPAI virus where taxa are represented by nodes and their relationships as edges (23). This corresponded very closely to the reassortants distinguished by BEAST analysis. The reassortants formed four groups: group I, first detected in May 2016; group II, first detected in August 2016 and including the reassortant that caused the main epi-demic in wild birds and poultry in Europe; group III, first de-tected in December 2016 and including a change of the NA serotype from eight to five; and group IV, also first detected in December 2016 (Figs. 3 and 4 and Table 1).
Spatiotemporal Distribution of the Main Reassortants. The clade H5N8 HPAI virus was widespread in Eurasia in 2016 and ex-tended into North Africa (Fig. 5). During 2016 and 2017, the virus spread widely from east Asia (SI Appendix, Table S1) westward to Europe, eastward to north Asia, and southward to south Asia and Africa between the second quarter (3-mo divi-sion) of 2016 and the second quarter of 2017. In the second quarter of 2016, the virus was detected sporadically in north-central China (Qinghai Lake) and the border between Russia and Mongolia (Uvs-Nuur Lake) (SI Appendix, Fig. S1). The westward spread to Europe started with infrequent detections in south-central Russia (Chany Lake and Kurgan in western Sibe-ria) in the third quarter of 2016 (SI Appendix, Fig. S2) and west Russia (Tatarstan) in the fourth quarter of 2016 (SI Appendix,
Fig. S3). Subsequently, the virus was detected at great frequency
in all parts of Europe (north, south, east, west, central) (SI
Ap-pendix, Table S1) in the fourth quarter of 2016 and the first
quarter of 2017 (SI Appendix, Fig. S4), followed by infrequent detections in the second quarter of 2017 (SI Appendix, Fig. S5). The virus also was detected in North Africa (Egypt) and south Asia (India) in the fourth quarter of 2016, in central Africa (Congo) in the second quarter of 2017, in north Asia (Kam-chatka, Russia) in the fourth quarter of 2016, and in east Asia (South Korea) from the fourth quarter of 2016 to the second quarter of 2017. The species affected were primarily wild birds in the second and third quarters of 2016, both wild birds and poultry in the fourth quarter of 2016 and first quarter of 2017, and poultry in the second quarter of 2017 (SI Appendix, Figs.
S1–S5). There was no apparent spatial association between the
distribution of sampled viruses and the density of the poultry population in Eurasia (SI Appendix, Fig. S6). During the period 2016/2017, H5N8 HPAI viruses were not detected in other parts of the world, including North and South America.
Inferred Hosts, Dates, and Locations of Origin of the Seven Main Reassortants.
Reassortant 1 (CABAD8AA; n= 16).This is the most ancestral reas-sortant in our analyses. The origin dates are estimated to be November 2015 for the most recent common grand ancestor (MRCGA) and February 2016 for the most recent common ancestor (MRCA) (SI Appendix, Fig. S7). The gene segments are inferred to originate from west, south, and east China for the MRCGA and north China for the MRCA. The inferred hosts of nearly all gene segments of both the MRCGA and MRCA are either long-range anseriform migrants or wild anseriforms. The exception is gene segment 6 (NA), for which the inferred hosts are domestic anseriforms. These dates and locations suggest that the MRCA of CABAD8AA originated at the end of the win-tering period 2015/2016 or beginning of spring migration 2016, with gene segment 6 contributed by domestic anseriforms from east China and remaining gene segments contributed by wild anseriforms (including long-range migrants) from wintering
MIC
locations in west, south, and east China. It cannot be ruled out that some gene segments (e.g., 3 and 7) were carried farther south during autumn migration 2015 to unsampled wintering locations in south Asia and then back to north China during spring migration.
The first actual CABAD8AA detections were from May 2016 on Qinghai Lake in China and Uvs-Nuur Lake at the Mongolian–Russian border (24, 25), just north of the median inferred location of origin of the MRCA. This suggests that this reassortant was carried farther north during spring migration
2016, presumably by wild birds on the way to breeding locations in north Russia. The first detected virus (A/Brown-headed_Gull/ Qinghai/ZTO1-LU/2016) had an HPAI virus H5 clade 2.3.4.4b-derived backbone of segments 4, 6, and 8, combined with seg-ments 1, 2, 3, 5, and 7 from different Asian low pathogenic avian influenza (LPAI) viruses (24, 25). After May 2016, this reas-sortant was not detected until May 2017, when it reemerged in domestic ducks in the Democratic Republic of Congo (26). Reassortant 2 (CAEAF8AA; n= 13).The new gene segments in this reassortant, compared with CABAD8AA, are segments 3 (PA) A B C D E F G 0 Group N1 N2 N3 N4 N5 N6 N7 N8 N-type N9 Africa Western Europe Northern Europe Southern Europe Eastern Europe Western Asia Northern Asia Southern Asia Region Eastern Asia Segment 1 (PB2) 2008.11 2013.00 2013.87 2016.51 2016.50 2015.69 2016.26 1 2 3 4 5 6 7 8 R 2010.66 2014.54 2013.48 Segment 2 (PB1) 2016.73 2017.06 2015.38 2016.17 1 2 3 4 5 6 7 8 R Segment 3 (PA) 2011.05 2012.93 2013.88 2016.42 2013.44 2015.85 2016.36 1 2 3 4 5 6 7 8 R 2016.73 Segment 4-H5 (HA) 1 2 3 4 5 6 7 8 R 2015.54 2016.26 2016.44 2016.44 2016.57
Fig. 1. Bayesian time-resolved phylogenetic trees for gene segments 1 to 4 of H5NX HPAI viruses sampled between May 2016 and July 2017 and for which
whole genomes were sequenced. Distinct clades (groups) in the trees are given different colored branches. The number of distinct groups differs per gene segment: four for segment 1, three for segment 2, five for segment 3, and one for segment 4. Furthermore, all eight segments of each virus are represented as eight parallel bars at the tips of each tree, with the colors in each segment’s bar corresponding to the branches of the individual gene sequences to indicate reassortment. The final bar represents the geographic locations of the sequences in the tree.
and 5 (NP). The inferred hosts of these new gene segments could be wild anseriforms, long-range anseriform migrants, other wild birds, or domestic anseriforms (SI Appendix, Fig. S8). The origin dates are estimated to be May 2016 for the MRCGA and August 2016 for the MRCA. The gene segments are inferred to originate from almost the full breadth of Eurasia (west Poland to east China) for the MRCGA and from a nar-rower breadth of Eurasia (east Azerbaijan to west China, but most locations in the region centered on the Kazakhstan–China
border) for the MRCA. These dates and locations suggest that the MRCA of CAEAF8AA originated during premigratory aggregation or autumn migration in the Kazakhstan–China border region.
The first actual CAEAF8AA detection (A/gadwall/Chany/97/ 2016) was from September 2016 in Russia. It was later found in wild birds at widely dispersed locations: from Italy to Korea and from Russia to Egypt and India. Except for Russia, these sites might be wintering locations of wild migratory birds.
Segment 7 (M) 1 2 3 4 5 6 7 8 R 2015.76 2016.35 2016.41 2016.63 2016.30 2016.22 2016.60 Segment 8 (NS) 1 2 3 4 5 6 7 8 R 2015.47 2016.33 2016.44 2016.45 2016.06 2016.21 2016.51 2015.76 2016.57 2016.60 Segment 6-N5 (NA) 1 2 3 4 5 6 7 8 R 2010.66 2016.34 Segment 6-N8 (NA) 1 2 3 4 5 6 7 8 R 2015.80 2016.34 2016.44 2016.47 2016.27 2015.91 2016.52 2016.00 2016.53 A B C D E F G 0 Group N1 N2 N3 N4 N5 N6 N7 N8 N-type N9 Africa Western Europe Northern Europe Southern Europe Eastern Europe Western Asia Northern Asia Southern Asia Region Eastern Asia Segment 5 (NP) 2011.10 2013.49 2014.16 2015.05 2015.24 2016.40 2015.27 1 2 3 4 5 6 7 8 R 2014.60 2016.31 2016.93 2015.79
Fig. 2. Bayesian time-resolved phylogenetic trees for gene segments 5 to 8 of H5NX HPAI viruses sampled between May 2016 and July 2017 and for which
whole genomes were sequenced. For segment 6, there are separate phylogenetic trees for subtypes N8 and N5. Distinct clades (groups) in the trees are given different colored branches. The number of distinct groups differs per gene segment: five for segment 5, two for segment 6, and one for segments 7 and 8.
Furthermore, all eight segments of each virus are represented as eight parallel bars at the tips of each tree, with the colors in each segment’s bar
corre-sponding to the branches of the individual gene sequences to indicate reassortment. The final bar represents the geographic locations of the sequences in the tree.
MIC
Reassortant 3 (AAAAA8AA; n= 109).This is the main virus detected in the 2016 to 2017 epidemic in Europe. The new gene segments in this reassortant, compared with CAEAF8AA, are segments 1 (PB2), 3 (PA), and 5 (NP). The inferred hosts of these new gene segments are long-range anseriform migrants (segment 1), other wild birds (segment 3), or either of these two groups (segment 5)
(SI Appendix, Fig. S9). The origin dates are estimated to be June
2016 for the MRCGA and July 2016 for the MRCA. The gene segments are inferred to originate from Belarus to India for the MRCGA and from a much more restricted area (Belarus to west Kazakhstan) for the MRCA. These dates and locations suggest that the MRCA of AAAAA8AA originated during premigratory aggregation or early autumn migration 2016 somewhere between Belarus and Kazakhstan.
The first actual AAAAA8AA detection (A/gadwall/Kurgan/ 2442/2016) was on 27 August 2016 in central Russia at the lon-gitude of the Ural Mountains. The second detection occurred on 2 October 2016 in Tatarstan, Russia, several hundred kilometers westward. From November 2016 onward, this virus was detected in wild birds in coastal regions of the Baltic and North Seas, as well as in wild birds on Lake Constance, Lake Biel, and Lake Neuchâtel, farther south in Europe. It also was found in wild birds in Ukraine, south Russia (Krasnodar), Hungary, and Italy. This suggests that the virus was carried west during autumn migration of wild birds in 2016, on the way to wintering locations in central, west, and south Europe.
Throughout Europe, this virus caused both massive die-offs in wild birds and outbreaks in poultry farms, lasting until at least summer 2017 (8, 20, 27–33).
Reassortant 4 (AADAA8AA; n= 14).The new gene segment in this reassortant, compared with AAAAA8AA, is segment 3 (PA). The inferred hosts of this gene segment are other wild birds (SI
Appendix, Fig. S10). The origin dates are estimated to be August
2016 for the MRCGA and October 2016 for the MRCA. The gene segments of the MRCA are inferred to originate from Germany to west Russia. These dates and locations suggest that the MRCA of AADAA8AA originated during late autumn migration 2016.
The actual detections of AADAA8AA were first in the Neth-erlands in November 2016 and then in Kaliningrad Oblast, Russia, in February 2017. This suggests that, following its generation in autumn 2016, this reassortant was restricted to a limited number of wintering locations in central and west Europe.
Reassortant 5 (DCBAE5AA; n= 15).The new gene segments in this reassortant, compared with CABAD8AA, are segments 1 (PB2), 5 (NP), and 6 (NA). The inferred hosts of these gene segments are wild anseriforms (segments 1 and 5), with ancestry of seg-ment 6 remaining unclear, although segseg-ment 6 clustered most closely with those of LPAI viruses of the subtypes H9N5 or H7N5 found in 2015 in shorebirds in Asia (A/common redshank/ Singapore/F83-1/2015, KU144675; A/black-tailed godwit/Ban-gladesh/24734/2015, KY635758) (SI Appendix, Fig. S11). The origin dates are estimated to be June 2016 for the MRCGA and July 2016 for the MRCA. The gene segments are inferred to originate from west-central Russia to northwest China to Thailand for the MRCGA and from south-central Russia for the MRCA. These dates and locations suggest that the MRCA of DCBAE5AA origi-nated during the breeding season or premigratory aggregation 2016. The actual detections of DCBAE5AA were in Georgia, the Czech Republic, Italy, and Germany from December 2016 to February 2017, suggesting that the reassortant was carried by wild birds during autumn migration 2016 to wintering locations in Europe. This virus caused outbreaks in poultry farms in Croatia and Germany. The reassortant also was detected in eastern Eurasia—on the Kamchatka Peninsula, Russia—in October 2016, which it likely reached via a different migration route.
Reassortant 6 (BABAB8AA; n= 56).This is the second most common virus detected during the 2016 to 2017 epidemic in Europe, after AAAAA8AA. The new gene segments in this reassortant, compared with CABAD8AA, are segments 1 (PB2) and 5 (NP). The inferred host of segment 1 is unclear (either domestic anseriforms or other wild birds), and the inferred host of seg-ment 5 is domestic anseriforms (SI Appendix, Fig. S12). The origin dates are estimated to be July 2016 for the MRCGA and September 2016 for the MRCA. The gene segments are inferred to originate from Ukraine to west Russia for the MRCGA and from several hundred kilometers due west—Hungary to Ukraine—for the MRCA. These dates and locations suggest that the MRCA of BABAB8AA originated during autumn migration 2016.
The first actual detections of BABAB8AA were in Croatia, Hungary, and France from October 2016. This virus was later detected farther north, in Germany and Poland in winter 2016 to 2017 (20, 33). This suggests that the reassortant spread in a restricted part of Europe during late autumn migration and the wintering period and potentially involved spillback of gene segments from domestic anseriforms to wild birds, possibly in Hungary.
Table 1. The names of the main reassortants and the phylogenetic groups within each gene segment
Reassortant
Date of first detection (full-genome sequence)
Estimated date of origin and 95% highest posterior density CIs
No. of sequences
Phylogenetic group per gene segment
1 2 3 4 5 6 7 8
No. Name Group PB2 PB1 PA HA NP NA M1 NS1
1 CABAD8AA I 5/1/16 3 February 2016 16 C A B A D 8 A A 11/15–4/16 2 CAEAF8AA I 9/10/16 9 August 2016 13 C A E A F 8 A A 5/16–9/16 3 AAAAA8AA II 8/27/16 13 July 2016 109 A A A A A 8 A A 5/16–8/16 4 AADAA8AA II 11/11/16 3 October 2016 14 A A D A A 8 A A 8/16–10/16
5 DCBAE5AA III 10/1/16 26 July 2016 15 D C B A E 5 A A
5/16–9/16
6 BABAB8AA IV 10/19/16 7 September 2016 56 B A B A B 8 A A
7/16–10/16
7 BDBAB8AA IV 12/2/16 4 November 2016 6 B D B A B 8 A A
Reassortant 7 (BDBAB8AA; n= 6). The new gene segment in this reassortant, compared with BABAB8AA, is segment 2 (PB1). The inferred hosts of this gene segment are wild anseriforms or long-range anseriform migrants (SI Appendix, Fig. S13). The origin dates are November 2016 for both the MRCGA and the MRCA. However, gene segment 2 has an earlier origin date, May 2016, suggesting that it reached west Europe in a two-step process: transport to breeding location in Siberia during spring migration 2016 and transport to wintering location in west Europe during
autumn migration 2016. The gene segments of the MRCGA are inferred to originate from a restricted region—Germany to Ukraine—except for gene segment 2, from west China; those of the MRCA are inferred to originate from an even more restricted region, Poland to Ukraine. These dates and locations suggest that the MRCA of BDBAB8AA originated during late autumn mi-gration 2016 or wintering period 2016 to 2017.
The actual detections of BDBAB8AA were first in Poland and Germany, from December 2016 onward. This suggests that the
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26312 0__A _turk ey_German y-NI _AR11550-L02126_ 2016__H5N8 __20 16 -Dec -29 300 743__A_ mallard _Poland_ 17_ 201 7__ H5N8__20 17- Jan-13 26 205 9__A_greylag_goose _German y-NI_AR703 -L02 13 8_2017_ _H5N8 __ 2017-Jan-2 5 26694 4__A_emu_Germa ny -ST_R321_2017__H 5N8_ _2017 -Jan-03 28 0672__A_buzzard_German y-ST_AR 1959 -L023 05 _20 17_ _H5N8__2017 -Feb-28 262058 __A_greyla g_goose_ German y-NI_AR1395-L02144_2 017__H5N8_ _201 7-F eb-02 30 0699 __A _domestic_goose_P oland_69 _20 17__ H5N8__2 017 -Jan-27 247 429__A_ domesti c_duck_Germa ny-BB_R 681ff _20 17_ _H5N8 __2 017 -Jan-26 29 1103__A_mute_s w an_German y_HE _AR195_20 17 __H5N8__2 017 -Jan 277 13 7__A_ sw an_German y-ST_AR 1166 -L02295_20 17 __H5N 8__2 01 7-F eb-02 26694 5__A_greyla g_goose_ Germa ny -SN_R513_ 201 7__H5N8_ _2017-Jan-21 300 70 1__A _tur key_ Poland _94_ 20 17_ _H5N8 __2017-F eb-03 24 010 2__A_domestic_goose _P ol and_3 3_2016__H5N 8__2016-De c-02 300686__A _turk ey_P oland _63 _20 16__ H5N8_ _2 016 -De c-12 288 737 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_yl at I _ no e gi w _ A _ _ 5 8 0 3 4 2255189__A_gadw al l_It aly_ 1 7VIR 133-2_201 7 __H5N5_ _ 20 1 7 -Jan-10 240 101__A_ mute_sw an_C roat ia_ 102 _20 16__H5N 5__2 016 -De c-2 7 261 332 __A _ chick en_Croat ia_1 04_ 201 7__ H5N5 __20 17-Ma r-07 243 698 __A _mute_s w an_Croatia_ 42_ 20 17__ H5N5 __2 017 -Jan-2 0 243 05 0__A _barna cle_goose_Ge rma n y-SH_R 115 05_ 2 016 __H5 N5__ 201 6-Dec -22 260 059 __A_ cormo rant_Ger man y-SH_R8 9 6_2 017 __H 5N5_ _20 17-Jan-3 0 24304 9__A_ turk ey_German y-SH_R4 25_20 17_ _H5N5__2 0 17-Jan-2 2 287564 __A _ T_D k_N L-We rkendam_ 1601415 9-0 01_201 6__H5N5 __20 16-N ov-1 4 274 858 __A_ mute_sw an_ Kal ining rad_1 32_20 17_ _H5N8__2 017-F eb-13 26865 9__A_Gull 1_N L-Mark er_W adden_ 160 144 66-011 _20 16__H5N8__2 016 -N ov-17 26866 9__A _T_ Dk_NL -Monn icke ndam_1 601386 5-0 06-0 08_ 2016_ _H5N8 __2 016 -Nov -08 26866 3__A_ Magpi e_N L-Volendam_16014 331-00 2_20 16_ _H5 N8__20 16-N ov-16 268 661__A_L -bl-ba-gull_N L-S ov on_16014324-014_201 6__H 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66-020 _2016_ _H5N8_ _2016 -Nov -17 268621__A_C_Gull_NL -Slootd orp_16014102-0 03_2 016__ H5N8_ _2016-N ov-11 268 619__A_ Bl_H _gull_ NL-Slootdorp_ 1601 410 2-002_ 2016__H5N8 __2 016-No v-11 268630__A _Dk_N L-Bidd inghuiz en_16015083 -01 6-02 0_2 016__H5N8 __2016-Nov -30 268629__A_Dk_NL -Bidd inghui zen_16014829 -011-015_2016__H5N8__20 16-No v-25 26863 1__A_ Dk_N L-Bidd inghuiz en_1 601 5145-02 1-0 25_ 201 6__ H5N8__2016-D ec-01 268 670__A_T_Dk_NL -Roggebotsl uis_16014462-015_ 2016_ _H5N8_ _2016-N ov-17 26867 7__A _T_ Dk_N L-Zeewolde_160 139 76-004 -00 6_2 016 __H5N 8__ 2016-No v-09 268678__A_T_Dk_NL -Zeewolde_160 13976-005 _20 16_ _H5N 8__2 016-Nov-09 241249 __A _domesti c_duck_Ge rman y-MV_ R9869_201 6__H 5N8__ 2016-N ov-23 24969 1__A _chic ken_German y-MV_R100 48_2016__H5N8_ _2016-No v-27 25681 8__A_domestic_duck_Ger man y-MV_R97 64_ 2016__H 5N8_ _2016-N ov-21 23803 9__A_chic ken_Germa ny-SH_R8758 _2016_ _H5N8 __2016 -Nov -11 238038__A_chicken_Germany-MV_R8790_2016__H5N8__2016-Nov-11 256817__A _turk ey_German y-NI _R98 07_2016 __H5N8__20 16-N ov-22 266 946__A_egret_Germa ny-SH_R1 315 _201 7__H5N 8__ 2017 -Ma r-11 255892__A_ Great _Blac k-back ed_Gull _Nethe rlands_ 2_2016__ H5N8__2016-N ov-23 257 699__A_T ufted_ Duck_S witzerland_V237_2 016__H5 N8__2016-No v-13 26866 2__A_M_S wan_N L-Roggebotsluis_ 1601 4462-0 19_2016_ _H5N8 __2016-Nov-17 23943 6__A_tufted_duck_S witzerland_V237-L02003_2016 __H 5N8_ _2016-Nov 300745 __A _wild_ duck_P oland_57_2017__ H5N8__2017- Jan-27 237958__A_ tufted_duck _Germa ny-SH_R8444_2016_ _H5N 8__2 016-N ov-07 237944 __A_tufted_duck_German y_AR8 444-L01 987_2016__H5N8__2016-N ov-07 24771 3__A_ tufted_duck _Denmark_17740 -1_20 16__ H5N8 __20 16-N ov-08 238037__A_ turkey_Germa ny-SH_R8 595_2016_ _H5N8 __201 6-Nov -09 23773 3__A _tufted_duck_German y_AR8444 -L01986 _2016__H5N8 __20 16-No v-07 237945__A _tufted_duck _Germa ny_AR8 459-L01 988_2016__ H5N8_ _2016-No v-08 23943 4__A_ tufted_duck _Swit zerland_V24 6-L020 01_2016 __H5 N8__201 6-Nov 240111__A_herring_gull_P oland_84_201 6__H5N8 __2016-No v-09 23943 7__A _caspian_gull_ Switzerland_V2 47-L020 04_201 6__H5N8__20 16-No v 268668__A _T_Dk_N L-Almeerder_Z and_ 16014341-003_2016__ H5N8 __20 16-No v-16 268679__A_ T_Dk_NL -Zeewolde_ 1601397 6-006_ 2016__H5N8 __2016-Nov-09 268676__A_T_ Dk_NL -Zeewolde_16013976-004_20 16__H5N8 __2016 -Nov-09 26867 5__A_T_ Dk_NL -Zeewolde_1601 3976-001 -003_2 016__H5N8__ 2016-N ov-09 26867 4__A_T_ Dk_NL-Z eewolde_160139 76-001 _2016_ _H5N8 __2016 -Nov-09 26868 1__A_T_Dk_NL -Zuidoost_Beemster _16014148-009_201 6__H5N8_ _2016-N ov-14 26868 0__A_T_ Dk_NL-Zuidoo st_Beemster_1 601414 8-002_ 2016__H5 N8__20 16-No v-14 238897__A_Common_Goldene ye_Sweden_SV A16111 7KU0322_SZ0002165_201 6__H5N8 __2016-Nov -14 288747__A _tufted_duck_ Germa ny-RP_AR1181_2 017__H5 N8__ 2017-Feb 288734__A_ mute_s wan_German y-NW_AR1 08_201 7__H5N8_ _20 17-Jan 255209__A_Common _tern_Hungary_8187_2017 __H5N8__2017-Feb-28 268635 __A_Eur_W ig_NL-Akkrum_1 601581 7-003_ 2016__ H5N8__20 16-Dec-13 268682__A _Teal_N L-Ferwer t_16015273 -013_2016__H 5N8__2016-Dec -01 268640__A_ Eur_Wig_NL -Ferwert_ 16015273-002_20 16__H5N 8__2016-De c-01 268660 __A_Gull1 0_NL-Mark er_Wadden_1601 4466-014 _2016__H5N8_ _2016-No v-17 268624__A_Ch_NL -Den_Oev er_160142 31-001_ 2016__H 5N8__2016-No v-15 268618__A_ Bk_swan_NL -Den_Oev er_16013973-002_2016__H5N8_ _20 16-Nov-10 268667__A_Sea_e agle_NL -Assen_16015398-002_2016__H5N8 __2016-Dec -05 268625__A _Ch_NL-Hiaure_ 16016112-001 -005_2016__ H5N8__2016-De c-17 268644__A_Eur_W ig_NL-Leeuw arden_16015699-002_2016__ H5N8__201 6-Dec-10 268647__A_ Eur_Wig_NL -Terschelling_ 16015692-0 10_2016__ H5N8__2016 -Dec-09 268671__A_ T_Dk_NL-Rotter dam_1601415 5-001_2016__ H5N8__2016-N ov-14 268633__A_Dk_N L-Rotterdam_1601400 8-001-005_2016__ H5N8__2016-N ov-10 238896__A_Chic ken_Sweden_SV A161122KU0453 _SZ0209321_ 2016__H5N8__2016 -Nov-21 238893__A_Chicken_S weden_SVA161122KU0 453_SZ0209316_2 016__H5N8__2016-No v-21 238894__A_Chick en_Sweden_SVA161122 KU0453_SZ0209317_ 2016__H5N8__2016-N ov-21 238895__A_Chicken_Sweden_SV A161122KU0453_SZ0209318_ 2016__H5N8__2016-Nov -21 263099__A_domestic_goo se_Germany-NI_AR51 9-L02153_2017__H5N8__2017-Jan-23 237921__A_wild_duck_P oland_82A_2016__H5N8__ 2016-Nov-02 239435__A_tufted_duck_S witzerland_V254-L02002_2016__H 5N8__2016-Nov 300747__A_mute_swan_Poland_137_2017_ _H5N8__2017-Feb-21 262057__A_mute_swan_Germany-NI_AR1529-L0 2145_2017__H5N8__2017-Feb-14 255910__A_Mew_Gull_Netherlands_1_2016__H5N8__2016-N ov-23 263117__A_turkey_Germany-NI_AR11365-L02128_2016__H5N8__2016-Dec-24 263119__A_turkey_Germany-NI_AR11461-L02127-40_2016__H5N8__2016-Dec-27 100.0 Group II Group I Group IV Group III 268639__A_Eur_Wig_NL-Enumatil-Groningen_16015704-001_2016__H5N8__2016-Dec-11 268672__A_T_ Dk_N L-W erk endam_160 14159-0 02_20 16_ _H5N8 __2 016 -No v-14 #7 BDBAB8AA #4 AADAA8AA #5 DCBAE5AA #6 BABAB8AA #3 AAAAA8AA #1 CABAD8AA #2 CAEAF8AA
Fig. 3. Supernetwork of 2016/2017 H5 HPAI viruses, based on full-genome sequences, generated by using maximum-likelihood (ML) phylogenetic trees of
sorted sequences according to segments. The eight ML trees of the segments were used to calculate a supernetwork. Each reassortant is indicated by a different color and assembled into four groups. The seven most common reassortants are numbered 1 to 7. Less common reassortants (total: four) are in-dicated in gray. Each circle represents full genome of HPAI virus, and the edges represent their phylogenetic relationship. Details as names, collection dates,
and coordinates are given inSI Appendix, Table S8.
MIC
reassortant originated locally in Europe due to a reassortment of its predecessor, BABAB8AA, with a gene segment from a wild anseriform, likely a long-range migrant from Siberia. This virus caused outbreaks in poultry farms in Poland, Germany, and Russia.
Summary of the Genesis and Spread of Seven Main Reassortants.In summary (Table 2 andSI Appendix, Fig. S13 and Table S2), most of the new gene segments for the reassortant viruses originated from wild birds (wild anseriforms, including long-range anseri-forms, or other wild birds), and the dates and locations of origin correspond with areas used by wild birds during different phases of their migratory cycles: wintering period to spring migration in north China, Mongolia, and Russia (Siberia) for reassortant 1; breeding period to autumn migration in Belarus/Kazakhstan/ China/Russia for reassortants 2, 3, and 5; autumn migration in Hungary/Germany/Ukraine/Russia for reassortants 4 and 6; and autumn migration to wintering period in Poland/Ukraine for reassortant 7. The only new gene segments not thought to originate from wild birds were gene segment 6 of reassortant 1 and gene segment 5 of reassortant 6; these gene segments were inferred to originate from domestic anseriforms.
Migratory Patterns of Wild Birds Found Positive for H5N8 HPAI Virus in the European Union.Based on reports to the Office Interna-tional des Epizooties (OIE), H5N8 HPAI viruses were detected in a total of 56 species found dead in the European Union be-tween 1 October 2016 and 5 July 2017 (18). Of these 56 species, 14 species had migratory populations that wintered in the Eu-ropean Union and bred at longitudes at least 60°E, which is at
the longitude of the Ural Mountains (Table 3). Of these 14 species, 13 were water birds belonging to the family Anatidae (including ducks, geese, and swans), while 1 species belonged to the family Turdidae (thrushes).
Discussion
The overall impact of the 2016/2017 epidemic of H5N8 HPAI virus (belonging to cluster B, Gochang like) differed markedly from the 2014/2015 epidemic of H5N8 HPAI virus (belonging to cluster A, Buan/Donglim like), both in geographical focus and the breadth of the host species affected. The 2016/2017 epidemic caused the largest recorded HPAI epidemic in poultry in Europe (18, 36) but did not spread to North America or Japan. In contrast, the 2014/2015 epidemic resulted in only limited poultry mortality in Europe and Japan (3) but caused major losses in the United States and to a lesser extent, in Canada, with over 48 million heads of poultry lost or destroyed (37). During the 2016/ 2017 epidemic, there was high mortality in free-living wild birds in Europe. In the Netherlands alone, ∼13,600 wild birds of 71 species were found dead during the epidemic (8). Considering an estimated detection rate of only 10 to 25%, the actual mortality probably was much higher and represented substantial percent-ages of wintering populations of several species in the Nether-lands: 5% of tufted ducks and Eurasian wigeons, 2 to 10% of greater black-backed gulls, and 11 to 39% of peregrine falcons (8). Also during the 2016/2017 epidemic, H5N8 HPAI virus caused unusually high mortality in white-tailed eagles, with 17 laboratory-confirmed fatal infections in Germany between November 2016 and April 2017 (9), and in a well-monitored
Fig. 4. Overview of the main reassortments described in this manuscript. Group designations and colors correspond to those in Fig. 3. The positions of the
population of mute swans (C. olor) in the United Kingdom, causing an age-adjusted mortality of 143 per 1,000 birds (38). In most cases, the effect of this mortality of wild birds at the pop-ulation level was not clear. However, the mortality rate of tufted ducks in parts of the Netherlands was so high (estimated at 25% of the local population) that population dynamics might have been affected substantially (8). In contrast, high wild bird mor-tality in Europe was not recorded in 2014/2015 (3). In the United States in 2014/2015, the majority of wild ducks did not show clear clinical signs of disease, although there was high mortality of wild raptor species (eagles, hawks, falcons, and owls) and wild geese (39). Experimental infections confirmed that the 2016/2017 H5N8 HPAI virus was substantially more virulent in domestic ducks than that from 2014/2015 (40).
We speculate that the high virulence of the H5N8 HPAI virus in 2016/2017 may be a side effect of selection for high excretion in wild water birds. Higher virus excretion not only results in more efficient transmission among hosts but also in increased severity of clinical signs. According to the intermediate virulence (or trade-off) hypothesis, there is selective advantage for higher excretion up to the point that higher transmission is counter-acted by increased clinical signs and even death (41). It is at this point of intermediate virulence that the virus has the greatest
evolutionary fitness. This fitness level depends in part on the level of population immunity, which will depend on the pro-portion of naïve birds and the presence of other circulating strains (38). It remains to be seen whether virulence of the H5N8 HPAI virus will change as it further evolves within the interna-tional metapopulation of wild birds and poultry. It also remains to be seen how the zoonotic potential of the H5N8 HPAI virus will change; based on experimental ferret infections with three of the viruses discussed here, the adaption of H5N8 HPAI virus to avian hosts was associated with a reduced zoonotic potential (40, 42).
The range of wild bird species involved in the 2016/2017 epi-demic in Europe differed from that in 2014/2015, although nonuniform sampling over time and space cannot be ruled out as a potential bias. In 2014/2015, only four wild bird species were found positive for H5N8 in Europe. In contrast, 56 species were found positive in Europe in 2016/2017 (18). Of these 56 species, 14 had migratory populations that wintered in the European Union and bred at longitudes at least 60°E (Table 3). These 14 species included the same species as found in 2014/2015 (Eur-asian wigeon, common teal, mallard, and mute swan) but also, 10 others. Thus, in 2016/2017 there was evidence for many more wild bird species that could have transported H5N8 viruses from
A
B
C
Fig. 5. Geographical distribution of detected H5NX HPAI viruses with full-genome sequences between the second quarter of 2016 and the second quarter of
2017. Viruses were detected from the east coast of Asia to the northwest coast of Europe, southern to northern Africa, and in south Asia. A–C show magnified
maps of (A) the northwest coast of Europe, (B) southern Africa, and (C) the east coast of Asia. Lambert conformal conic projection. Light green: reassortant 1; dark green: reassortant 2; red: reassortant 3; purple: reassortant 4; orange: reassortant 5; light blue: reassortant 6; dark blue: reassortant 7; circle: Wild-ans;
square: Wild-ans-long; pentagon: Wild-other; rhomboid: Dom-ans; triangle: Dom-gal. Map image credit: Copyright © 1995–2020 Esri. All rights reserved.
Published in the United States of America.
MIC
breeding areas in Siberia to wintering areas in Europe. Which of these species transport virus remains to be determined and re-quires more detailed investigation of the candidate hosts, both in the context of avian influenza epidemiology and long-range movement ecology.
Reassortment frequency was much higher in the 2016/2017 epidemic than in the 2014/2015 epidemic and than in the Eu-ropean epidemic of H5N1 HPAI virus in 2006/2007. In 2016/ 2017, 11 reassortants were detected (Fig. 3), of which 7 were common (Fig. 4 and Table 1). In 2014/2015, three reassortants were detected in North America (10) and none elsewhere (3). For the reassortment of gene segments from two influenza viruses to occur, both viruses need to infect the same host cell at the same time. Possible reasons for the high frequency of reas-sortment in 2016/2017 may be related to the extent and timing of the epidemic, the host range infected by H5N8 HPAI virus, target tissues infected by H5N8 virus, and the ease with which the H5N8 HPAI virus reassorts with other influenza viruses (e.g., due to special features of some gene segments). The extent of the epidemic in wild birds was much greater in Europe in 2016/ 2017 than in 2014/2015 and may explain the higher frequency of
reassortants in 2016/2017. Similarly, the fact that the 2014/2015 epidemic was greater in the United States than in Europe may explain the occurrence of reassortants in the United States (10, 43). Based on estimated MRCAs, the epidemic in Europe in 2016/2017 (MRCA of most common reassortant 3: July) started 1 mo earlier than in 2014/2015 (MRCA for HA and NA: Au-gust), so it may have coincided better with the peak of LPAI virus infection in wild water birds in autumn (44), thereby in-creasing the chance of reassortment between H5N8 HPAI virus and LPAI virus. The likelihood of the two peaks coinciding de-pends on how long it takes a virus to reach peak prevalence after its appearance in a wild waterfowl population. Although the host range of wild water birds infected with H5N8 HPAI virus in Europe in 2016/2017 was larger than in 2014/2015, dabbling duck species, particularly common teal and mallard, were found in-fected with H5N8 HPAI virus in both epidemics (Table 3). These latter species are considered to have the highest prevalence of LPAI virus infection (45) and therefore, considered to be im-portant “mixing vessels” between HPAI virus and LPAI virus. Based on experimental infections of Pekin ducks, the H5N8 HPAI virus of 2016/2017 had greater tropism for the small Table 2. Overview of spatiotemporal spread of reassortants of H5 HPAI viruses in the 2016/2017 epidemic from time of origin of MRCA to 1 July 2017
No. Reassortant
Time of origin of MRCA
Country of origin of MRCA
Host origin of new gene segments
Subsequently detected spread of virus Month in 2016 Phase of life cycle* Europe† Northern Africa Southern Africa‡ South Asia East Asia
1 CABAD8AA February M, W China Not relevant X X
3 AAAAA8AA July M, B Belarus to
Kazakhstan
Wild bird X
5 DCBAE5AA July M, B Russia Wild bird X X
2 CAEAF8AA August M Azerbaijan to China Not determined X X X X
6 BABAB8AA September M, W Hungary to Ukraine Wild bird and poultry X
4 AADAA8AA October M, W Germany to Russia§ Wild bird X
7 BDBAB8AA November M, W Poland to Ukraine Wild bird X
B, breeding; M, migration; W, wintering; X, virus detected. *Phase of life cycle of long-distance migratory birds breeding in Siberia.
†Ural Mountains taken as geographical border between Europe and Asia.
‡South border of Sahara taken as geographical border between northern and southern Africa.
§Kaliningrad.
Table 3. Wild bird species in which H5N8 HPAI virus was detected in dead birds found in the European Union between 1 October 2016 and 5 July 2017 and reported to the OIE and in which at least some populations migrate long distance
Species migrating long distance*
No. of H5N8-positive dead birds
Estimated
population size Ref.
Common name Scientific name
Mute swan† C. olor 1,217 495,000 34, p. 46
Tufted duck Aythya fuligula 190 1,800,000 34, p. 187
Whooper swan Cygnus cygnus 149 57,000 34, p. 51
Eurasian wigeon† M. penelope 89 1,810,000 34, p. 117
Mallard† Anas platyrhynchos 55 3,250,000 34, p. 132
Greater white-fronted goose Anser albifrons 15 1,365,000 34, p. 66
Common pochard Aythya ferina 10 1,700,000 34, p. 177
Common teal† A. crecca 7 2,960,000 34, p. 123
Common shelduck Tadorna tadorna 2 155,000 34, p. 101
Lesser white-fronted goose Anser erythropus 2 63,000 34, p. 70
Red-crested pochard Netta rufina 2 250,000 34, p. 171
Bewick’s swan Cygnus bewickii 2 17,000 34, p. 55
Common goldeneye Bucephala clangula 1 420,000 34, p. 217
Common fieldfare Turdus pilaris 1 42,850,000 35
*Here defined as wintering in the European Union and breeding at least beyond longitude 60°E, at the level of the Ural Mountains.
