Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice

Since mid-2016,the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic(HP)phenotype in China,raising many concerns about public health and poultry industry.The insertion of a“KRTA”motif at hemagglutinin cleavage site(HACS)occurred in the early stage of HP H7N9 variants.During the co-circulation,the HACS of HP-H7N9 variants were more polymorphic in birds and humans.Although HP-H7N9 variants,unlike the H5 subtype virus,exhibited the insertions of basic and non-basic amino acids,the underlying function of those insertions and substitutions remains unclear.The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China.Then,we generated six H7N9 viruses bearing the PEIPKGR/G,PEVPKGR/G,PEVPKRKRTAR/G,PEVPKGKRTAR/G,PEVPKGKRIAR/G,and PEVPKRKRR/G motifs.Interestingly,after the deletion of threonine and alanine(TA)at HACS,the H7N9 viruses manifested decreased thermostability and virulence in mice,and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence.By contrast,the insertion of non-basic amino acid isoleucine and alanine(IA)decreased the transmissibility in chickens and virulence in mice.Remarkably,the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens,suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice.The ongoing evolution of H7N9 increasingly threatens public health and poultry industry,so,its comprehensive surveillance and prevention of H7N9 viruses should be pursued.

Neutrophil-derived PAD4 induces citrullination of CKMT1 exacerbates mucosal inflammation in inflammatory bowel disease

Peptidyl arginine deiminase 4(PAD4)plays a pivotal role in infection and inflammatory diseases by facilitating the formation of neutrophil extracellular traps(NETs).However,the substrates of PAD4 and its exact role in inflammatory bowel disease(IBD)remain unclear.In this study,we employed single-cell RNA sequencing(scRNA-seq)and substrate citrullination mapping to decipher the role of PAD4 in intestinal inflammation associated with IBD.Our results demonstrated that PAD4 deficiency alleviated colonic inflammation and restored intestinal barrier function in a dextran sulfate sodium(DSS)-induced colitis mouse model.scRNA-seq analysis revealed significant alterations in intestinal cell populations,with reduced neutrophil numbers and changes in epithelial subsets upon PAD4 deletion.Gene expression analysis highlighted pathways related to inflammation and epithelial cell function.Furthermore,we found that neutrophil-derived extracellular vesicles(EVs)carrying PAD4 were secreted into intestinal epithelial cells(IECs).Within IECs,PAD4 citrullinates mitochondrial creatine kinase 1(CKMT1)at the R242 site,leading to reduced CKMT1 protein stability via the autophagy pathway.This action compromises mitochondrial homeostasis,impairs intestinal barrier integrity,and induces IECs apoptosis.IEC-specific depletion of CKMT1 exacerbated intestinal inflammation and apoptosis in mice with colitis.Clinical analysis of IBD patients revealed elevated levels of PAD4,increased CKMT1 citrullination,and decreased CKMT1 expression.In summary,our findings highlight the crucial role of PAD4 in IBD,where it modulates IECs plasticity via CKMT1 citrullination,suggesting that PAD4 may be a potential therapeutic target for IBD.