2.2. Generation of IGIP-H1 Influenza Plasmids

The IGIP protein is highly conserved among mammals and expressed by antigen-presenting dendritic cells (DCs) in the intestinal tract as a 47–54 aa protein. IGIP is thought to play a role in the regulation of IgA expression in the intestinal tract. The C-terminal 24 aa in IGIP correspond to the mature active peptide, whereas the N-terminal ~30 aa correspond to the signal peptide region (Figure 1A). DNA fragments with the sequence corresponding to the 5′ untranslated region (UTR) and signal peptide sequence of H1 HA (A/California/04/09 (Ca/04) (H1N1)), followed by a G4S linker, furin cleavage site, the Thosea assigna virus (TAV) 2A protease and the mature IGIP were generated with a cloning spacer downstream and acquired from Genscript (Piscataway, NJ, USA). The fragment was digested with AarI (Thermo Scientific, Waltham, MA, USA) and cloned into the reverse genetic plasmid pDP2002 as previously described, generating an intermediate plasmid pDP2002-IGIP [15,16]. The pDP2002-IGIP was digested with BsmBI (New England BioLabs, Ipswich, MA, USA) and the HA Ca/04, previously amplified by PCR using Phusion High-fidelity PCR master mix with GC buffer (Thermo Scientific) was cloned into the pDP2002-IGIP generating the plasmid pDP2002-IGIP-H1. The pDP2002-IGIP-H1 sequence was confirmed by Sanger sequencing (Psomagen, Rockville, MD, USA).

In vitro growth kinetics. (A) Alignment of the predicted IGIP in different mammalian species. The mature swine IGIP sequence used in this study is shown. (B) Schematic representation of the IGIP-H1 gene. The IGIP sequence and the components of the intergenic region are indicated. (C) Growth kinetics profiles of IGIP-H1att and H1att in MDCK and MDCK STAT1 KO cells. Experiments were performed two times independently, each time in triplicate. Titers were determined by RT-qPCR and expressed as log₁₀ TCID50 equivalents. Gray area represents the area below the level of detection of the assay. (D) IGIP-H1att virus was serially passaged five times in MDCK (E1C5) cells and SPF eggs (E5), and the HA was amplified by RT-PCR, showing that the IGIP-H1 rearrangement is stable. Abbreviations: FP, fusion peptide; TM, transmembrane domain; CT, c-terminal region; G4S, poly-glycine protein linker; Furin CS, furin cleavage site; Tav2A, Thosea assigna virus 2A protein sequence; GlucS, Signal peptide of Gaussia luciferase.