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Genetic diversity of highly pathogenic avian influenza a(H5N8/H5N5) viruses in Italy, 2016-17.

In spring 2016, highly pathogenic avian influenza (HPAI) outbreaks caused by the H5N8 subtype of clade 2.3.4.4 (group B) were reported in migratory wild birds in Qinghai Lake, China (1), and in the salt lake system of Uvs Nuur on the Russian Federation-Mongolia border (2). Since then, HPAI A(H5N8) viruses have been detected in several countries in Asia, Europe, and Africa. In Europe, the virus was detected for the first time in October 2016 in Hungary (3). Here, we describe the occurrence of multiple introductions of reassortant HPAI A(H5N8) and A(H5N5) viruses in Italy, in both wild and domestic birds.

The Study

During December 2016-January 2017, a Eurasian wigeon (Anas penelope) and agadwall (Anasstrepera) found dead at Grado Lagoon in northeastern Italy tested positive for HPAI A(H5N5). A second wigeon tested positive for HPAI A(H5N8). Since then, additional HPAI A(H5N8) cases were observed in a common shelduck (Tadorna tadorna) and in a mute swan (Cygnus olor) and in birds on 6 commercial turkey farms, 1 layer farm, and 3 backyard flocks (Table 1; Figure 1). All of the cases in domestic poultry farms occurred in areas in close proximity to wetlands that are listed as important resting sites for migratory waterfowl. The onset of clinical signs in all the affected poultry species was generally associated with depression, reluctance to move, and a drop in feed consumption. The clinical condition often evolved into a more severe respiratory and nervous syndrome associated with an increased mortality rate (average mortality rate is 1.62% [95% CI 1.10%-2.14%]). Depopulation measures on the infected farms and 7 neighboring poultry premises considered at risk involved [approximately equal to] 510,000 birds.

The genomes of 10 positive samples collected from wild (n = 4) and domestic (n = 6) birds were fully sequenced (online Technical Appendix 1, https://www.nc. cdc.gov/EID/article/23/9/17-0539-Techappl.pdf). Phylogenetic analysis of the hemagglutinin (HA) gene showed that the HPAI A(H5N5) and A(H5N8) viruses clustered within the 2.3.4.4 clade, group B (Figure 1). However, the characterization of the complete genome (online Technical Appendix 1 Figures 1-8) revealed that these viruses belong to 4 distinct genotypes, which had very likely originated from multiple reassortment events.

Phylogenetic analyses indicated that the HPAI H5N5 viruses had been generated through intersubtype reassortment events between the H5N8 viruses from Asia (H5N8Gs/Qinghai/2016-like) and the low pathogenicity avian influenza (LPAI) viruses of the Eurasian lineage (Figure 2). The A(H5N8) viruses from Asia were the source of the HA, polymerase acidic, matrix, and nonstructural protein genes. HPAI A(H5N5) viruses with similar HA and neuraminidase genes were identified in Croatia and Czech Republic in 2016-17. The time to the most recent common ancestor (tMRCA) estimated by pooling the information across all the gene segments in a hierarchical model (online Technical Appendix 1) suggested that a virus with this gene constellation emerged during October-December 2016 (Table 2; online Technical Appendix 1 Table 1).

Among the 8 HPAI A(H5N8) viruses in Italy investigated during this study, 5 were collected from wild and domestic birds in the Veneto region. In all the phylogenetic trees, these viruses clustered within the main European A(H5N8) group (A/wild duck/Poland/82A/ 2016-like) (Figure 2), previously described by Pohlmann et al. (4). The tMRCA for this group was May-June 2016 in the hierarchical gene segment model (Table 2; online Technical Appendix 1 Table 1). The first HP AI A(H5N8) virus detected in a turkey farm in the Veneto region displayed the gene composition of a virus isolated in October 2016 from a painted stork in an Indian zoo (5), which had not previously been reported in Europe (Figure 2). The tMRCA of this Indian-Italian group is July-October 2016, according to the hierarchical gene segment model (Table 2; online Technical Appendix 1 Table 1). The 2 outbreaks reported in 2 commercial turkey farms in the Emilia-Romagna and Lombardy regions were caused by HP AI A(H5N8) reassortant viruses containing the polymerase basic protein 2 and nucleoprotein genes of LP AI viruses of the Eurasian lineage and the remaining genes from the H5N8-Gs/Qinghai/2016-like genotype (Figure 2). Viruses with a similar gene pool were identified in Croatia and France. Estimation of the tMRCA by the hierarchical gene segment model indicated that this genotype might have emerged during June-August 2016 (Table 2; online Technical Appendix 1 Table 1).

Analyses of the phylogenetic topologies revealed that most of the sequences found in Italy were dispersed throughout the trees, indicating the occurrence of several independent introductions of the A(H5N8) virus into poultry farms from wild birds (online Technical Appendix 1 Figures 1-8). These results were confirmed by our median-joining network analyses for the HA gene (online Technical Appendix 1 Figure 9), which showed that the ancestral sequences of the samples from Italy represent viruses collected in other countries. In most cases [greater than or equal to] 1 median vector, representing the lost ancestral sequences, separated these viruses from the hypothetical progenitor. The only exception was for A/turkey/Italy/17VIR576-11/2017 and A/turkey/Italy/17VIR1452-22/2017, which proved to be almost identical for all the genes (similarity of 99.9%-100%), although they were collected 24 days apart in 2 turkey flocks located at a distance of [approximately equal to] 90 km from one another and no evident contacts were observed between them. However, because the 2 outbreaks had occuned in 2 farms operated by the same company, an exchange of virus cannot be ruled out.

Intravenous pathogenicity indexes obtained for 8 representative A(H5N8) and A(H5N5) isolates ranged from 2.85-3, comparable to an index of 2.93 for 2016 A(H5N8) viruses from Gennany and 2.75-2.84 for 2016 A(H5N8) viruses from Russia (2,4). These data confirm that both of the A(H5N8) and A(H5N5) viruses from Italy, which shared the same HA cleavage site (PLREKRRKR), are highly pathogenic for poultry.

Conclusions

Since its emergence in China in 2013, the HP AI H5 of clade 2.3.4.4 has evolved in different genetic groups, namely A to D (6). Here, we describe the introductions of 4 different H5 viral genotypes of clade 2.3.4.4 group B in northern Italy. As previously observed for the 2014-15 A(H5N8) epidemic wave (7), our results confirm that these strains have a high propensity to reassort with co-circulating LP AI and HP AI viruses, causing the generation of several subtypes and genotypes with unique gene constellations. Unfortunately, the lack of sequences of the potential progenitors, exemplified by the long branches observed in particular in the polymerase basic protein 2, polymerase acidic, and nucleoprotein phytogenies, makes it difficult to determine when and where these genotypes emerged. The genetic variability observed in the viruses identified in domestic birds. the similarity to viruses circulating in Europe and India, and the close proximity of the infected poultry farms to wetlands all suggest that wild birds did play a major role in the multiple and independent introductions of the virus into poultry holdings.

Our study highlights the importance of generating complete viral genome sequences in a timely fashion, which may help to monitor the viral spread and define appropriate disease control strategies. This, coupled with intensified wild bird surveillance on wetlands of ecologic importance for avian influenza viruses, can improve our understanding of the virus dissemination routes and support early detection of viruses highly pathogenic to poultry or believed to be of immediate concern to human health.

EID Podcast: Novel Eurasian Highly Pathogenic Avian Influenza A H5 Viruses in Wild Birds, Washington, USA, 2014

The novel Eurasian lineage clade 2.3.4.4 highly pathogenic avian influenza (HPAI) A(H5N8) virus spread rapidly and globally during 2014, substantially affecting poultry populations. The first outbreaks were reported during January 2014 in chickens and domestic ducks in South Korea and subsequently in China and Japan, reaching Germany, the Netherlands, and the United Kingdom by November 2014 and Italy in early December 2014. Also in November 2014, a novel HPAI H5N2 virus was reported in outbreaks on chicken and turkey farms in Fraser Valley, British Columbia, Canada. This H5N2 influenza virus is a reassortant that contains the Eurasian clade 2.3.4.4 H5 plus 4 other Eurasian genes and 3 North American wild bird lineage genes. Taiwan has recently reported novel reassortants of the H5 clade 2.3.4.4 with other Eurasian viruses (H5N2, H5N3).

The appearance of highly similar Eurasian H5N8 viruses in Asia, Europe, and now the United States suggests that this novel reassortant may be well adapted to certain waterfowl species, enabling it to survive long migrations. These appearances also represent a major change in Eurasian H5 virus circulation. After the reported spread of HPAI H5N1 virus in Asia, a large, interagency avian influenza virus (AIV) surveillance effort was implemented throughout the United States during April 2006-March 2011. Of nearly 500,000 wild bird samples tested, none harbored Eurasian subtype H5 AIV. The overall prevalence of AIV was 11%, and most viruses (86%) were detected in dabbling ducks (family Anatidae). Although H5N8 subtype viruses have been detected previously in the United States, all have been low pathogenicity AIV of North American wild bird lineage.

DOI: https://doi.org/ 10.3201/eid2309.170276

Acknowledgments

We acknowledge the authors and the originating and submitting laboratories of the sequences from the GISAID EpiFlu Database on which this research is based in part (online Technical Appendix 2, https://www.nc.cdc.gov/EID/ article/23/9/17-0539-Techapp2.xlsx). We also thank Annalisa Salviato, Alessia Schivo, Sabrina Marciano, and Francesca Ellero for their excellent technical assistance.

This study was financially supported by the European project PREDEMICS (research project supported by the European Community's Seventh Framework Programme [FP7/2007-2013] under grant agreement 278433) and the Italian Ministry of Health (RC IZSVe 05/14).

Dr. Fusaro isa biotechnologist working at the Istituto Zooprofilattico Sperimentale delle Venezie. Her primary research interests include molecular epidemiology, intra- and inter-host evolution, gene flow, and cross-species transmission of RNA viruses.

References

(1.) Li M. Liu H. Bi Y. Sun J. Wong G. Liu D. et at. Highly pathogenic avian influenza A(H5N8) viras in wild migratory birds, Qinghai Lake, China. Emerg Infect Dis. 2017; 23:637-41. http://dx.doi.org/10.3201/eid2304.161866

(2.) Lee DH, Sharshov K, Swayne DE, Kurskaya O, Sobolev I, Kabilov M, et al. Novel reassortant clade 2.3.4.4 avian influenza A(H5N8) virus in wild aquatic birds, Russia, 2016. Emerg Infect Dis. 2017; 23:359-60. http://dx.doi.org/10.3201/eid2302.161252

(3.) World Organisation for Animal Health. Updated on highly pathogenic avian influenza in animals (type H5 and H7), 2016 [cited 2017 Mar 10]. http://www.oie.int/animal-health-in-theworld/update-on-avian-influenza/2016

(4.) Pohlmann A, Starick E, Harder T, Grand C, Hoper D, Globig A et al. Outbreaks among wild birds and domestic poultry caused by re-assorted influenza A(H5N8) clade 2.3.4.4 viruses, Germany, 2016. Emerg Infect Dis. 2017; 23:633-6. http://dx.doi.org/10.3201/eid2304.161949

(5.) Nagarajan S, Kumar M, Muragkar HV. Tripathi S, Shukla S, Agarwal S, et al. Novel reassortant highly pathogenic avian influenza (H5N8) virus in zoos, India. Emerg Infect Dis. 2017; 23:717-9. http://dx.doi.org/10.3201/eid2304.161886

(6.) Lee DH. Bahl J. Torchetti MK. Killian ML. Ip HS. DeLiberto TJ. et al. highly pathogenic avian influenza viruses and generation of novel reassortants, Linited States, 2014-2015. Emerg Infect Dis. 2016; 22:1283-5. http://dx.doi.org/10.3201/eid2207.160048

(7.) Hall JS, Dusek RJ, Spackman E. Rapidly expanding range of highly pathogenic avian influenza viruses. Emerg Infect Dis. 2015; 21:1251-2. http://dx.doi.org/10.3201/eid2107.150403

Alice Fusaro, [1] Isabella Monne, [1] Paolo Mulatti, Bianca Zecchin, Lebana Bonfanti, Silvia Ormelli, Adelaide Milani, Krizia Cecchettin, Philippe Lemey, Ana Moreno, Paola Massi, Tiziano Dorotea, Stefano Marangon, Calogero Terregino

Author affiliations: Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy (A. Fusaro, I. Monne, P. Mulatti, B. Zecchin, L. Bonfanti, S. Ormelli, A. Milani, K. Cecchettin, T. Dorotea, S. Marangon, C. Terregino); Rega Institute, KU Leuven, Leuven, Belgium (P. Lemey); Istituto Zooprofilattico Sperimentale delia Lombardia e deN'Emilia Romagna, Brescia, Italy (A. Moreno, P. Massi).

[1] These authors contributed equally to this article.

Address for correspondence: Alice Fusaro, Istituto Zooprofilattico Sperimentale delle Venezie, Research & Innovation Department, OIE and National Reference Laboratory for avian influenza & Newcastle disease, FAO Reference Centre for animal influenza and Newcastle disease, OIE Collaborating Centre for Diseases at the Human-Animal Interface, Viale dell'Universita 10, 35020, Legnaro, Padua, Italy; email: afusaro@izsvenezie.it

Caption: Figure 1. Highly pathogenic avian influenza A(H5N8) and A(H5N5) in birds, Italy, 2016-17). A) Geographic distribution of cases in wild (red) and domestic (blue) birds in northern Italy. Squares indicate the samples sequenced in this study; circles indicate positive samples for which no genetic information was available at the time of writing. B) Maximum-likelihood phylogenetic tree of the hemagglutinin gene of clade 2.3.4.4 viruses. Viruses analyzed in this study are indicated with red (wild birds) and blue (domestic birds) squares, numbered according to the collection date. Bootstrap supports >60% are indicated above the nodes. Scale bar indicates nucleotide substitutions per site.

Caption: Figure 2. Probable genesis of highly pathogenic avian influenza A(H5N8) and A(H5N5) reassortant viruses identified in Italy, 2016-17 (gray box). Virus particles are represented by ovals containing horizontal bars that represent the 8 gene segments, colored according to their origin.
Table 1. Epidemiologic information for highly pathogenic avian
influenza A(H5N5) and A(H5N8) viruses isolated from birds in Italy,
2016-17

Isolate                            Type   Collection date

A/wigeon/Italy/16VIR9616-3/2016    H5N5     2016 Dec 29

A/wigeon/Italy/17VIR57-3/2017      H5N8     2017 Jan 03
A/gadwall/Italy/17VIR133-2/2017    H5N5     2017 Jan 10
A/swan/Italy/17VIR537-2/2017       H5N8     2017 Jan 19
A/turkey/Italy/17VIR538-1/2017     H5N8     2017 Jan 20
A/turkey/Italy/17VIR576-11/2017    H5N8     2017 Jan 23
A/chicken/Italy/17VIR653-12/2017   H5N8     2017 Jan 25
A/turkey/Italy/17VIR973-2/2017     H5N8     2017 Feb 01
A/turkey/Italy/17VIR1338-3/2017    H5N8     2017 Feb 14
A/turkey/Italy/17VIR1452-22/2017   H5N8     2017 Feb 16

Isolate                                   Region

A/wigeon/Italy/16VIR9616-3/2016    Friuli Venezia Giulia

A/wigeon/Italy/17VIR57-3/2017      Friuli Venezia Giulia
A/gadwall/Italy/17VIR133-2/2017    Friuli Venezia Giulia
A/swan/Italy/17VIR537-2/2017       Friuli Venezia Giulia
A/turkey/Italy/17VIR538-1/2017            Veneto
A/turkey/Italy/17VIR576-11/2017           Veneto
A/chicken/Italy/17VIR653-12/2017          Veneto
A/turkey/Italy/17VIR973-2/2017        Emilia Romagna
A/turkey/Italy/17VIR1338-3/2017          Lombardy
A/turkey/Italy/17VIR1452-22/2017          Veneto

Isolate                                   Location

A/wigeon/Italy/16VIR9616-3/2016        Grado (Gorizia)

A/wigeon/Italy/17VIR57-3/2017          Grado (Gorizia)
A/gadwall/Italy/17VIR133-2/2017        Grado (Gorizia)
A/swan/Italy/17VIR537-2/2017          Aquileia (Udine)
A/turkey/Italy/17VIR538-1/2017          Mira (Venice)
A/turkey/Italy/17VIR576-11/2017    Piove di Sacco (Padua)
A/chicken/Italy/17VIR653-12/2017     Porto Viro (Rovigo)
A/turkey/Italy/17VIR973-2/2017         Sorbolo (Parma)
A/turkey/Italy/17VIR1338-3/2017     Monzambano (Mantova)
A/turkey/Italy/17VIR1452-22/2017   Gazzo Veronese (Verona)

Isolate                                  Site type

A/wigeon/Italy/16VIR9616-3/2016         Natural park

A/wigeon/Italy/17VIR57-3/2017           Natural park
A/gadwall/Italy/17VIR133-2/2017         Natural park
A/swan/Italy/17VIR537-2/2017            Natural park
A/turkey/Italy/17VIR538-1/2017     Fattening turkeys farm
A/turkey/Italy/17VIR576-11/2017    Fattening turkeys farm
A/chicken/Italy/17VIR653-12/2017      Laying hens farm
A/turkey/Italy/17VIR973-2/2017     Fattening turkeys farm
A/turkey/Italy/17VIR1338-3/2017    Fattening turkeys farm
A/turkey/Italy/17VIR1452-22/2017   Fattening turkeys farm

Isolate                                EpiFlu
                                   accession no. *

A/wigeon/Italy/16VIR9616-3/2016     EPI888600-01,
                                    EPI954800-05
A/wigeon/Italy/17VIR57-3/2017       EPI888085-92
A/gadwall/Italy/17VIR133-2/2017     EPI954616-23
A/swan/Italy/17VIR537-2/2017        EPI954552-59
A/turkey/Italy/17VIR538-1/2017      EPI954560-67
A/turkey/Italy/17VIR576-11/2017     EPI954568-75
A/chicken/Italy/17VIR653-12/2017    EPI954576-83
A/turkey/Italy/17VIR973-2/2017      EPI954584-91
A/turkey/Italy/17VIR1338-3/2017     EPI954592-99
A/turkey/Italy/17VIR1452-22/2017    EPI954600-07

* GISAID EpiFlu database (http://platform.gisaid.org).

Table 2. tMRCA for the 4 avian influenza A(H5N5) and A(H5N8)
virus genotypes identified in Italy, 2016-17 *

                                                  tMRCA

Genotype                                          Mean

H5N5                                          November 2016
H5N8 A/wild duck/Poland/82A/2016-like           May 2016
H5N8 A/painted stork/India/10CA03/2016-like    August 2016
H5N8 A/mute swan/Croatia/70/2016-like           July 2016

                                                      tMRCA

Genotype                                             95% HPD

H5N5                                          October-December 2016
H5N8 A/wild duck/Poland/82A/2016-like             May-June 2016
H5N8 A/painted stork/India/10CA03/2016-like     July-October 2016
H5N8 A/mute swan/Croatia/70/2016-like           June-August 2016

* tMRCAs estimated for each gene segments are reported in online
Technical Appendix 1 Table 1 (https://www.nc.cdc.
goV/EID/article/23/9/17-0539- Techappl .pdf).HPD, highest posterior
density; tMRCA, time to most recent common ancestor.
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Title Annotation:DISPATCHES
Author:Fusaro, Alice; Monne, Isabella; Mulatti, Paolo; Zecchin, Bianca; Bonfanti, Lebana; Ormelli, Silvia;
Publication:Emerging Infectious Diseases
Article Type:Report
Geographic Code:4EUIT
Date:Sep 1, 2017
Words:2800
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