Evolution of Highly Pathogenic Avian Influenza A(H5N1) Virus in Poultry, Togo, 2018

In 2015, highly pathogenic avian influenza A(H5N1) viruses reemerged in poultry in West Africa. We describe the introduction of a reassortant clade 2.3.2.1c virus into Togo in April 2018. Our findings signal further local spread and evolution of these viruses, which could affect animal and human health.


The Study
In April 2018, high mortality rates were reported in chickens (84%) and quails (27%) on a farm with 4,371 domestic birds and 89 swine in Lacs Province in the south of Togo. Necropsies revealed petechiae and hemmorrhages in tracheas, bursa of Fabricius, lungs, and livers. We suspected influenza A virus, which we subsequently confirmed by using the Flu Detect rapid test (Synbiotics Corporation, http://www.synbiotics.com) according to the manufacturer's instructions. We collected 15 samples, including cloacal and tracheal swab specimens and tissues (liver, trachea, lung, and spleen), from euthanized chickens with clinical signs and confirmed the presence of H5 by using reverse transcription PCR. As a precaution, all the animals on the farm (including swine) were slaughtered, even if clinical signs were observed only in birds. Humans in contact with animals on the farm were put under surveillance by public health services (no samples were collected but medical checkup was offered).
All the isolates from Togo were closely related to each other, which can be explained by the limited duration  (7,9), providing further support for Nigeria (and not Côte d'Ivoire) as a possible source of the Togo virus. Although most similar to sequences from viruses in Nigeria, the sequences from Togo displayed a degree of divergence (1.3% as calculated with the maximum composite distance) from their closest relatives as evidenced by tree topologies (Appendix Figures 1, 2).  Figure 3). Whether outbreaks went unnoticed or unreported in the region or whether specific selection pressures might have existed in Togo still requires further investigation. We observed higher genetic diversity among the sequences of the 2015-2016 isolates from Côte d'Ivoire than among the isolates from Togo. Most viruses from Côte d'Ivoire that we sequenced in this study belonged to the subcluster WA1 (on the basis of their HA and NA gene segments) and were closely related to viruses from Burkina Faso, Nigeria, and Ghana (closest strain was A/domestic bird/Burkina Faso/15VIR1774-22/2015) (Appendix Figures 1, 2). Two isolates from Côte d'Ivoire clustered with WA2 viruses from the same outbreak from Côte d'Ivoire and viruses from Nigeria in both HA and NA phylogenies (Appendix Figures 1, 2). We also identified WA1 HA and WA2 NA reassortants. Full genome sequencing of these isolates from Togo and Côte d'Ivoire is warranted to allow for a full assessment of recent genetic reassortments in the region.

Evolution of Highly Pathogenic Avian Influenza
On the basis of HA sequences, we estimated the tMRCA of the WA2 strains from Côte d'Ivoire (as well as those from the closely related strains previously re- . Although we performed limited (and not randomized) sampling, which probably yielded results that are not representative of the complete epidemiologic context, we nevertheless observed substantial virus diversity in the region.

Conclusions
After a period of absence, HPAI clade 2.3.2.1c H5 viruses have spread in sub-Saharan Africa in the past 3 years. A single introduction of virus into the region might have occurred, followed by local spread, leading to genetic and antigenic diversification. The high prevalence of these viruses in countries with large commercial poultry industries, such as Nigeria and Côte d'Ivoire, has resulted in reassortment between local viral lineages. Adding to the complexity, HPAI A(H5N8) viruses from clade 2.3.4.4 have recently been reported in Cameroon and Nigeria (2,12), and low-pathogenicity avian influenza H9N2 viruses have also spread from northern to western Africa in 2017-2018 (with a report from Burkina Faso) (13)(14)(15). If these viruses also establish endemicity, the avian influenza situation in western Africa will be in stark contrast to the situation over the past decade or more, when limited virus circulation occurred. The threat to animal and public health should therefore be seriously reconsidered, especially because veterinary services in the region might not operate at the efficiency required to quickly identify and contain outbreaks.

Acknowledgments
We gratefully acknowledge the originating and submitting laboratories of the sequences from the GISAID EpiFlu Database (http://www.gisaid.org), on which this research is based, and Kim Friedman for data management. We are grateful to the creators of the Genotoul bioinformatics platform Toulouse Occitanie for providing computing resources, Patrice Dehais for his help with Galaxy, and Christelle Camus-Bouclainville for her help with the figures.

About the Author
Mr. Fusade-Boyer is a PhD student at the UMR Interactions Hôtes Agents Pathogènes in Toulouse, France. His primary research interests include influenza viruses at the animalhuman interface.