Genetic characterization of highly pathogenic avian influenza H5N1 viruses isolated from naturally infected pigeons in Egypt Emad Mohamed Elgendy 1,2 • Yohei Watanabe 1,3 • Tomo Daidoji 1 • Yasuha Arai 1,3 • Kazuyoshi Ikuta 3 • Madiha Salah Ibrahim 1,2 • Takaaki Nakaya 1 Received: 23 March 2016 / Accepted: 23 June 2016 Ó Springer Science+Business Media New York 2016 Abstract Avian influenza viruses impose serious public health burdens with significant mortality and morbidity not only in poultry but also in humans. While poultry suscep- tibility to avian influenza virus infection is well charac- terized, pigeons have been thought to have low susceptibility to these viruses. However, recent studies reported natural pigeon infections with highly pathogenic avian influenza H5N1 viruses. In Egypt, which is one of the H5N1 endemic areas for birds, pigeons are raised in towers built on farms in backyards and on house roofs, providing a potential risk for virus transmission from pigeons to humans. In this study, we performed genetic analysis of two H5N1 virus strains that were isolated from naturally infected pigeons in Egypt. Genetic and phylogenetic analyses showed that these viruses originated from Egyp- tian H5N1 viruses that were circulating in chickens or ducks. Several unique mutations, not reported before in any Egyptian isolates, were detected in the internal genes (i.e., polymerase residues PB1-V3D, PB1-K363R, PA-A369V, and PA-V602I; nucleoprotein residue NP-R38K; and non- structural protein residues NS1-D120N and NS2-F55C). Our findings suggested that pigeons are naturally infected with H5N1 virus and can be a potential reservoir for transmission to humans, and showed the importance of genetic analysis of H5N1 internal genes. Keywords H5N1 Á Pigeon Á Egypt Á Sequencing Á Phylogenetic analysis Highly pathogenic avian influenza H5N1 (HPAI H5N1) viruses circulating in Egypt are classified in clade 2.2 [1]. These viruses have accumulated genetic mutations that allowed them to be classified as clade 2.2.1, which is unique to Egypt. Clade 2.2.1 viruses diverged thereafter in 2008–2009 into phylogenetically distinct sublineages that clustered into two clades [2–4]. The viruses in the one, clade 2.2.1-C viruses, have enhanced receptor binding affinity to human-type a2,6-linked sialic acid, indicating a possible higher transmission efficiency to humans. Viruses in the other, clade 2.2.1.1 viruses, retain receptor binding affinity to avian-type a2,3-linked sialic acid, but are anti- genically distinct from clade 2.2.1-C viruses. Among the two groups, clade 2.2.1-C has been dominant in Egypt since 2011, and was reclassified as clade 2.2.1.2 [2, 4]. These results suggested a phenotypic diversification of Egyptian H5N1 viruses with the potential to infect a new host(s). Therefore, Egypt is considered as one of the hot spots of H5N1 virus evolution [5], emphasizing the need to understand the dynamics of H5N1 virus spread and circu- lation among both domesticated and wild birds. Edited by William Dundon. Electronic supplementary material The online version of this article (doi:10.1007/s11262-016-1369-z) contains supplementary material, which is available to authorized users. & Yohei Watanabe nabe@biken.osaka-u.ac.jp & Takaaki Nakaya tnakaya@koto.kpu-m.ac.jp 1 Department of Infectious Diseases, Kyoto Prefectural University of Medicine, 465 Kawaramachi-hirokoji Kamigyo-ku, Kyoto 602-8566, Japan 2 Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhur University, Damanhur, Egypt 3 Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan 123 Virus Genes DOI 10.1007/s11262-016-1369-z