Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites

Abstract

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.

H9N2 Influenza Virus Infections in Human Cells Require a Balance between Neuraminidase Sialidase Activity and Hemagglutinin Receptor Affinity

“…In this study, after virus adsorption, the cells were washed and then overlaid with 1% methylcellulose in medium without trypsin to prevent the release and spread of progeny virus, thereby enabling us to observe only foci that were a result of the initial input virus infection. Most H9N2 G1 lineage viruses, including CL80, CL42, and Em/R66, had HAs with mono- or dibasic cleavage sites, and their growth depended on trypsin ( 20 , 39 ). The viruses showed identical infectivity on MDCK cells ( Fig.…”

A risk marker of tribasic hemagglutinin cleavage site in influenza A (H9N2) virus

“…This study demonstrated that during serial passaging in chickens, the P2 position at HACS of the H9N2 virus was more polymorphic, and the amino acids N337K in the P2 position detected in the 6th passage 33 . The G1-lineage H9N2 viruses were adapted to birds and humans and induced more severe infections than the other representative lineage of H9N2 virus, and a basic amino acid in the P3 position of HACS formed an intermediate step towards a gain in pathogenicity 34 . In this study, we found that a sharp increase occurred after 2010 in the G1-lineage of H9N2 viruses with an identical HACS of the tribasic PAKSKR/G-motif from Bangladesh.…”

A Semiquantitative Scoring System for Histopathological and Immunohistochemical Assessment of Lesions and Tissue Tropism in Avian Influenza

“…Results of the histopathological and immunohistochemical examination and the basic semiquantitative scoring have been published in multiple independent manuscripts for most of the infection experiments previously (see [39][40][41][42][43][44][45][46]).…”