In this study, HPAI (H5N8) virus, A/Goose/Iraq/Sul.1/2018, was detected in geese in Sulaymaniyah province in Kurdistan Region, Iraq. Phylogenetic analysis reveals that the virus fell in group B in clade 2.3.4.4 H5N8. The topology of the phylogenetic tree based on the HA gene indicated that the Iraq virus clustered with viruses isolated from Iran and Israel in 2016-2017. The topology of the phylogenetic tree based on the NA gene showed that the Iraq virus clustered with viruses in Iran and Belgium. According to both HA and NA clusters, A/Goose/Iraq/Sul.1/2018 has a common ancestor with A/Crow/Aghakhan/2017, which was isolated from the migratory hooded crow in a national park in Esfahan province of Iran [17].
Because Iraq and Iran are located in the path of Black Sea-Mediterranean flyways and West Asian-East African flyway of migratory bird [18], it was suggested that both A/Goose/Iraq/Sul.1/2018 and A/Crow/Aghakhan/2017 may have originated from the same source of migratory birds. Furthermore, according to previous database research on the transmission of H5N1 in Iraq, poultry trading is more likely associated with the transmission of avian influenza [18]. Iraq shares a long international border with Iran, and legal and illegal commercial and poultry trades take place between the two countries. Therefore, we cannot exclude that A/Goose/Iraq/Sul.1/2018 may have been transmitted from Iran.
According to the report of the World Organization for Animal Health (OIE), the first outbreak of H5N8 in Iraq was in January of 2018 [14]. However, the sequences of the viral HA and NA have not been identified previously. Hence, our study about the characterization of the Iraqi H5N8 is considered the first report about HPAI subtype H5N8 in geese in Iraq.
Unfortunately, few sequences of H5N8 were available in the GenBank databases from the Middle East and only 34 H5N8 isolates were submitted to GISAID across the globe, which hindered the analysis of the avian influenza virus isolate in this study.
Multiple insertions of basic amino acids at the cleavage site of the HA gene were major determinants of pathogenicity of the H5 virus [19]. The sequence of the HA gene of A/Goose/Iraq/Sul.1/2018 showed that the virus possessed the molecular markers for HPAI, with a polybasic amino acid cleavage site motif, PLREKRRKR/GLF. The receptor-binding site curtails the host range of influenza virus [20]. The single amino acid substitutions A132S, Q222 L, G224S, Q192H, Q192R, S223 N, and N220 K (H5 numbering) of HA protein have been reported to increase the affinity of avian influenza virus to from α-2,3 sialic acid (avian) to α-2, 6 sialic acid (human)[6, 21].
The receptor binding domains of A/Goose/Iraq/Sul.1/2018 were H103, E186, N189, K192, K189, G221, Q222, R223, and G224, which revealed the preference for classic avian α-2, 3 sialic acid specificity. In this study, Iraq HPAI subtype H5N8 had two substitutions in HA at S133A and T156A, like most H5N8 subtypes. These substitutions increase the affinity for α2,6 sialic acid receptors in mammals [22, 23]. In spite of that, according to OIE, there were no reported cases of human infection with H5N8 influenza virus so far [14].
Susceptibility of avian influenza to antiviral drugs is associated with the sequence characteristic of NA protein [24]. Previous studies showed that molecular markers of resistance to zanamivir are V116A, R118K, E119G/A/D, Q136K, D151E, R152K, E277D, R292K and oseltamivir resistance markers are I117V, E119V, D198N, H274Y, R292K, and N294S (N2 numbering) [25]. Analysis of NA-deduced amino acid of Iraq H5N8 showed that there were no markers for oseltamivir, zanamivir, and Peramivir. Therefore, A/Goose/Iraq/Sul.1/2018 may be susceptible to antiviral drugs that act via the inhibition of NA.