title: Characterization of clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses from wild birds possessing atypical hemagglutinin polybasic cleavage sites.
abstract: Since 2014, clade 2.3.4.4 H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have been distributed worldwide. These viruses, which were reported to be highly virulent in chickens by intravenous inoculation, have a consensus HPAI motif PLRERRRKR at the HA cleavage site. However, two-clade 2.3.4.4 H5N8 viruses which we isolated from wild migratory birds in late 2014 in Japan possessed atypical HA cleavage sequences. A swan isolate, Tottori/C6, had a novel polybasic cleavage sequence, PLGERRRKR, and another isolate from a dead mandarin duck, Gifu/01, had a heterogeneous mixture of consensus PLRERRRKR and variant PLRERRRRKR sequences. The polybasic HA cleavage site is the prime virulence determinant of AIVs. Therefore, in the present study, we examined the pathogenicity of these H5N8 isolates in chickens by intravenous inoculation. When 106EID50of these viruses were intravenously inoculated into chickens, the mean death time associated with Tottori/C6 was substantially longer (>6.1 days) than that associated with Gifu/01 (2.5 days). These viruses had comparable abilities to replicate in tissue culture cells in the presence and absence of exogenous trypsin, but the growth of Tottori/C6 was hampered. These results indicate that the novel cleavage motif of Tottori/C6 did not directly affect the infectivity of the virus, but Tottori/C6 caused attenuated pathogenicity in chickens because of hampered replication efficiency. It is important to test for the emergence of diversified HPAIVs, because introduction of HPAIVs with a lower virulence like Tottori/C6 might hinder early detection of affected birds in poultry farms.
authors.affiliation: The Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.|Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.
authors.forename: Tatsufumi
authors.initials: T
authors.lastname: Usui
authors.affiliation: The Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.|Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Soda
authors.affiliation: The Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.|Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Tomioka
authors.affiliation: The Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.|Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ito
authors.affiliation: Avian Influenza Research Centre, Kyoto Sangyo University, Kamigamo-motoyama, Kita, Kyoto, 603-8555, Japan.
authors.forename: Toshiyo
authors.initials: T
authors.lastname: Yabuta
authors.affiliation: Avian Influenza Research Centre, Kyoto Sangyo University, Kamigamo-motoyama, Kita, Kyoto, 603-8555, Japan.|Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita, Kyoto, 603-8555, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takakuwa
authors.affiliation: The Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.|Avian Influenza Research Centre, Kyoto Sangyo University, Kamigamo-motoyama, Kita, Kyoto, 603-8555, Japan.|Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita, Kyoto, 603-8555, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Otsuki
authors.affiliation: The Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.|Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Ito
authors.affiliation: The Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan. yamaguti@muses.tottori-u.ac.jp.|Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyama Minami, Tottori, 680-8553, Japan. yamaguti@muses.tottori-u.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamaguchi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000835:Animals, Wild / Q000821:virology; D002460:Cell Line; D002645:Chickens; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000071225:Influenza A Virus, H5N8 Subtype / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D005585:Influenza in Birds / Q000401:mortality / Q000821:virology*; D010802:Phylogeny; D011201:Poultry Diseases / Q000821:virology; D017422:Sequence Analysis, DNA; D019562:Viral Load; D014774:Virulence; D014779:Virus Replication
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus
keywords: Clade 2.3.4.4; Cleavage site; H5N8 subtype; Highly pathogenic avian influenza; Virulence; Wild bird