Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)-Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV.

Marcel Gischke,Elsayed M Abdelwhab,Thomas C Mettenleiter,David Scheibner,Eva Böttcher-Friebertshäuser,Beate Crossley,Olanrewaju I Fatola,Ahmed H Salaheldin,Jutta Veits,Reiner Ulrich

doi:10.3390/ijms21072353

Abstract

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Highlights

Influenza A viruses are members of the family Orthomyxoviridae and divided into equine, classical swine/human, gull, bat and avian influenza virus (AIV) lineages [1,2]
HPAIVs evolve from LPAIV of H5 or H7 subtype after the acquisition of a polybasic cleavage site (CS), which is specific in each HPAIV
We showed that furin-like protease(s) can cleave the HA of H4N2_T327K in transfected human embryonic kidney 293T (HEK293T) independent of the presence of Human airway trypsin-like protease (HAT) and transmembrane protease serine 2 (TMPRSS2) that activate some viruses with monobasic CS [33] and some H9N2 viruses with monobasic VSSR/G, dibasic RSSR/G or tribasic RSRR/G cleavage site motifs [32]

Summary

Introduction

Influenza A viruses are members of the family Orthomyxoviridae and divided into equine, classical swine/human, gull, bat and avian influenza virus (AIV) lineages [1,2]. HPAIVs evolve from LP progenitors after circulation in domesticated birds and are naturally restricted to H5 and H7 subtypes. The monobasic CS of LPAIV is activated by trypsin-like proteases, which are restricted to the respiratory and/or gastrointestinal tracts of birds resulting in only local infections. Human airway trypsin-like protease (HAT) and transmembrane protease serine 2 (TMPRSS2) present in human airways have been shown to cleave HA with a monobasic cleavage site. It remains to be investigated whether orthologous proteases (e.g., HAT and TMPRSS2) of birds support HA cleavage in birds. The polybasic CS of HPAIV is cleaved by ubiquitous, subtilisin-like proteases causing systemic infection and multiorgan dysfunction [9]

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