Effect of molecular determinants in the hemagglutinin gene on the emergence of H7N9 highly pathogenic influenza virus in China

Abstract

Low pathogenic avian influenza (LPAI) viruses of H5 and H7 subtypes possess the potential to evolve to the highly pathogenic (HP) form. HPAI acquires an insertion of a multi-basic amino acid motif at the HA cleavage site (CS), which expands the HA cleavability by ubiquitously expressed furin-like proteases and increases viral tissue tropism. China H7N9 LPAIs that cause zoonotic infections in humans since 2013 and evolved into HPAI in 2016 after a four-year-circulation among poultry in China. HA genes of H7N9 LPAIs can be phylogenetically classified into Yangtze River Delta (YRD) and Pearl River Delta (PRD) lineages, while H7N9 HPAIVs emerged from YRD-like viruses. The hypothesis is that specific molecular determinants in the HA gene of YRD-like viruses may have facilitated the insertion of the multi-basic amino acid motif at the HA CS.

Polymorphism at HA CS has been identified among H7N9 HPAI isolates, with two dominant sequences reported PKGKRTAR/G (motif-1) and PKRKRTAR/G (motif-4). Phylogenetic analyses suggested HPAI viruses containing motif-4 may have evolved from those containing motif-1. I investigated the HA cleavability of these two motifs in avian and mammal cell lines. In the absence of exogenous trypsin, HA cleavage of motif-1 was less efficient in human 293T cells but was efficient in chicken DF1 cells. In contrast, HA with motif-4 showed comparable cleavability in both cell lines without exogenous trypsin. Recombinant H7N9 virus carrying motif-4 also showed broader tissue tropism in mice and embryonated chicken eggs than the recombinant H7N9 virus carrying motif-1.

To study molecular determinants associated with the evolution of China H7N9 viruses from LPAI to HPAI, LPAI recombinant H7N9 viruses carrying consensus HA gene representing PRD-like or YRD-like viruses were constructed and serially passaged in embryonated chicken eggs or in chicken embryonic fibroblasts (CEF). Additional recombinant viruses were generated to study the effects of specific HA mutations (A122S and L226Q), or the effect of polymerase fidelity (PB1-V43I mutation) during sequential passages. In addition, the HA gene of an HPAI H7N9 virus was mutated into LPAI as a control virus. The mutations in the HA genes were monitored every five passages by next-generation sequencing (NGS). After 15 serial passages under a high multiplicity of infection (MOI) (107 M gene copies/egg) or after 10 serial passages under a low MOI (105 M gene copies/egg) in 14-day eggs, no insertion above the 1% detection threshold was found near the HA CS from any of the six recombinant viruses. No insertion was found after ten passages in CEF cells with limited exogenous trypsin. However, positive selection was found at the HA receptor-binding domain after serial passages in eggs or in CEF. The A160T (H3 numbering) mutation was commonly found in both egg-passaged and CEF-passaged viruses, while the L226Q (H3 numbering) was dominant in eggs passaged viruses.

These results provide a first assessment of the molecular differences between YRD and PRD HA genes on the emergence of the HP H7N9 viruses under laboratory settings, where the selection pressure is less stringent compared to the field settings.