Emerging influenza D viruses (IDVs), the newest member in the genus Orthomyxovirus family, which can infect and transmit in multiple mammalian species as its relatives the influenza A viruses (IAVs). Additional studies of biological characteristics of IDVs are needed; here, we studied the characteristics of IDV nonstructural protein 2 (NS2), which shares the lowest homology to known influenza proteins. First, we generated reassortant viruses via reverse genetics to analyze the segment compatibility and gene interchangeability between IAVs and IDVs. Next, we investigated the locations and exact sequences of nuclear export signals (NESs) of the IDV NS2 protein. Surprisingly, three separate NES regions were found to contribute to the nuclear export of an eGFP fusion protein. Alanine scanning mutagenesis identified critical amino acid residues within each NES, and co-immunoprecipitation experiments demonstrated that their nuclear export activities depend on the CRM1-mediated pathway, particularly for the third NES (136-146aa) of IDV NS2. Interestingly, the third NES was important for the interaction of NS2 protein with CRM1. The findings in this study contribute to the understanding of IDV NS2 protein’s role during nucleocytoplasmic transport of influenza viral ribonucleoprotein complexes (vRNPs) and will also facilitate the development of novel anti-influenza drugs targeting nuclear export signals of IDV NS2 protein.

The replication and transcription of influenza virus depends on the participation of many host factors in cells. Exploring the relationship between viruses and host factors will help us fully understand the characteristics and pathogenic mechanisms of influenza viruses.

MicroRNAs (miRNAs) are important regulators involved in the antiviral response to influenza virus infection, however, an analytical comparison of miRNA and mRNA expression changes induced by several H7N9 host-adapting PB2 mutants remains undone. Here, miRNA microarray and transcriptome sequencing of BALB/c mouse lungs infected with A/Anhui/1/2013 (H7N9) [hereafter referred to as H7N9/AH1-PB2-627K(WT)] and mutant variants with PB2 amino acid substitutions (avian-like H7N9/AH1-PB2-627E and mammalian-adapted H7N9/AH1-PB2-627E/701N) were directly compared. The results showed that influenza virus infection induced dysregulation of numerous host cell processes. In a miRNA-mRNA network associated with immunity, changes in the expression of 38 miRNAs and 58 mRNAs were detected following influenza virus infection. Notably, the miRNAs of mmu-miR-188-5p, mmu-miR-511-5p, mmu-miR-483-5p, and mmu-miR-690 were specifically associated with the replication of the avian-like virus H7N9/AH1-PB2-627E. Likewise, the miRNAs of mmu-miR-691, mmu-miR-329-3p, and mmu-miR-144-3p were specifically associated with the mammalian-adapted virus H7N9/AH1-PB2-627E/701N. Finally, the miRNAs of mmu-miR-98-5p, mmu-miR-103-3p, mmu-miR-199a-5p, and mmu-miR-378a-3p were specifically associated with H7N9/AH1-PB2-627K(WT) virus replication. This is the first report of comparative integration analysis of miRNA-mRNA expression of these three H7N9 influenza viruses with different host-adapting PB2 mutations. Our results highlight potential miRNAs of importance in influenza virus pathogenesis.

Influenza A viruses effectively hijack the intracellular "resources" to complete transcription and replication, which involve extensive interactions between the viral and host proteins. Herein, we screened the host factors, which belong to DExD/H-box protein family members, RNA-binding proteins or mitochondrial anchoring proteins, to investigate their effects on polymerase activity. We observed DDX39B and DDX39A, DEAD-box RNA-Helicases, exert a dual effect on regulating polymerase activity and replication of influenza A viruses. We further revealed that DDX39B and DDX39A interact with viral NP and NS1 proteins. Interestingly, the viral NP proteins could reverse the inhibitory effect of excess DDX39B or DDX39A on polymerase activity. Mechanistically, the TREX complex subunits, THOC1, THOC4 and CIP29, were recruited to DDX39B-DDX39A-NP complex in an ATP-dependent manner, via the interaction with DDX39B or DDX39A, followed by excess TREX-NP complexes interfere with the normal oligomerization state of NP depending on the ratio between the viral and host proteins. On the other hand, the TREX complex, an evolutionarily conserved protein complex, is responsible for the integration of several mRNA processing steps to export viral mRNA. Knockdown of TREX complex subunits significantly down-regulated viral titers and protein levels, accompanied by retention of viral mRNA in the nucleus. Taken together, screening the host factors that regulate the replication of influenza virus advances our understanding of viral pathogenesis and our findings point out a previously unclear mechanism of TREX complex function.

One avian H3N2 influenza virus, providing its PB1 and HA segments, reassorted with one human H2N2 virus and caused a pandemic outbreak in 1968, killing over 1 million people. After its introduction to humanity, the pandemic H3N2 virus continued adapting to humans and has resulted in epidemic outbreaks every influenza season. To understand the functional roles of the originally avian PB1 gene in the circulating strains of human H3N2 influenza viruses, we analyzed the evolution of the PB1 gene in all human H3N2 isolates from 1968 to 2019. We found several specific residues dramatically changed around 2002–2009 and remained stable through to 2019. Then, we verified the functions of these PB1 mutations in the genetic background of the early pandemic virus, A/Hong Kong/1/1968(HK/68), as well as a recent seasonal strain, A/Jiangsu/34/2016 (JS/16). The PB1 V709I or PB1 V113A/K586R/D619N/V709I induced higher polymerase activity of HK/68 in human cells. And the four mutations acted cooperatively that had an increased replication capacity in vitro and in vivo at an early stage of infection. In contrast, the backward mutant, A113V/R586K/N619D/I709V, reduced polymerase activity in human cells. The PB1 I709V decreased viral replication in vitro, but this mutant only showed less effect on mice infection experiment, which suggested influenza A virus evolved in human host was not always consisted with highly replication efficiency and pathogenicity in other mammalian host. Overall, our results demonstrated that the identified PB1 mutations contributed to the viral evolution of human influenza A (H3N2) viruses.

Background: Influenza virus remains a continuous and serious threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important. Methods: A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments.Results: The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433NAELLVL439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What’s more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy.Conclusions: Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

Background Influenza virus remains a continuous and severe threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important. Methods A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments. Results The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433NAELLVL439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What’s more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy. Conclusions Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.