An intravenous pathogenicity index (IVPI) of > 1.2 in chickens or, in case of subtypes H5 and H7, expression of a polybasic hemagglutinin cleavage site (HACS), signals high pathogenicity (HP). Viruses of the H9N2-G1 lineage, which spread across Asia and Africa, are classified to be of low pathogenicity although, in the field, they became associated with severe clinical signs and epizootics in chickens. Here we report on a pre-eminent trait of recent H9N2-G1 isolates from Bangladesh and India, which express a tribasic HACS (motif PAKSKR-GLF; reminiscent of an HPAIV-like polybasic HACS) and compare their features to H9Nx viruses with di- and monobasic HACS from other phylogenetic and geographic origins. In an in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, only tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses did not replicate in endothelial cells either in the chorio-allantoic membrane or in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity. Pathogenicity assessment of tribasic H9N2-G1 viruses remains problematic. It cannot be excluded that the formation of a third basic amino acid in the HACS forms an intermediate step towards a gain in pathogenicity. Continued observation of the evolution of these viruses in the field is recommended.

中文翻译：

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表达单，双或三碱基血凝素裂解位点的来自广泛地理区域的H9亚型禽流感野生型病毒的致病性比较

鸡的静脉内致病性指数（IVPI）> 1.2，或者在H5和H7亚型的情况下，表达多聚性血凝素裂解位点（HACS）表示高致病性（HP）。H9N2-G1谱系病毒遍布亚洲和非洲，被归类为低致病性，尽管在野外，它们与鸡的严重临床体征和流行病有关。在这里，我们报道了来自孟加拉国和印度的最新H9N2-G1分离株的杰出特征，它们表现出三元HACS（基元PAKSKR-GLF；让人联想到HPAIV样的多元HACS），并将它们的特征与带有di-以及其他系统发育和地理起源的一元HACS。在体外测定中 H9N2的三元HACS由类似于真正的H5 HPAIV的弗林蛋白酶样蛋白酶加工而成，而某些二元位点显示出可裂解性增加，而一元HACS没有。然而，所有病毒在细胞培养中仍然依赖胰蛋白酶。在卵内，仅三基H9N2病毒被发现在胚胎器官的广泛范围内复制。与所有亚型H5 / H7 HPAI病毒相反，三基H9N2病毒既不在绒毛膜尿囊膜中的内皮细胞中，也未在其他胚胎组织中复制。通过IVPI，所有H9Nx分离株均被证明具有低致病性。三基H9N2-G1病毒的致病性评估仍然存在问题。不能排除的是，HACS中第三种碱性氨基酸的形成是致病性增加的中间步骤。