Article Figures & Data
Figures
- FIG 1
Generation of infectious influenza D/swine/Oklahoma/1314/2011 virus by the reverse-genetics system. (A) Schematic representation of the bidirectional seven-plasmid-based reverse-genetics system for IDV. The seven IDV cDNA segments were cloned into the pHW2000 vector (31). Each of the cDNA segments was flanked by the human Pol I promoter and the murine terminator. The RNA Pol I transcription unit was flanked by the truncated CMV promoter and the bovine growth hormone (BGH) poly(A) signal. (B and C) Growth kinetics of the wild-type D/OK and the rescued D/OK-RGS viruses. MDCK cells were infected with the D/OK or D/OK-RGS virus at an MOI of 0.1 (B) or an MOI of 0.01 (C). Samples of supernatants were collected at the indicated times, and virus titers were then determined in MDCK cells by a TCID50 assay. The data presented in panels B and C are representative of results from three independent experiments, with each experiment analyzing samples in duplicate.
- FIG 2
Growth kinetics of continuously passaged influenza D/OK-RGS viruses at different temperatures. (A) MDCK cells were infected with D/OK-RGS virus at an MOI of 0.01. After 5 days of infection, culture supernatants were collected and used for the next passage in MDCK cells. Virus titers at the indicated passages were determined by a TCID50 assay. (B and C) MDCK cells were infected with different passages of D/OK-RGS virus at an MOI of 0.01, followed by further incubation at 37°C (B) or 33°C (C). Samples of supernatants were collected at the indicated days and then titrated by a TCID50 assay. The data presented are representative of results from three independent experiments, with each experiment analyzing samples in duplicate.
- FIG 3
Receptor-binding properties of the wild-type D/OK virus and the recombinant D/OK-RGS virus. (A) Chemical structures of sialic acids that serve as receptors for influenza viruses. (B and C) Inhibition of viral hemagglutination by receptor analogs. The receptor analog Neu5,9Ac2 (B) or Neu5Gc9Ac (C) at the indicated concentrations was added to the virus containing 4 HA units. Mixtures were incubated for 30 min at room temperature. Aliquots of turkey RBCs were then added to the mixtures, and results were read after 30 min at room temperature. PBS was used as a negative control. Note that “−” indicates no hemagglutination, while “+” denotes evident hemagglutination. The data presented in panels B and C are representative of results from four independent experiments, with each experiment analyzing samples in duplicate.
- FIG 4
Activity of the IDV RNP complex with the PB1-E697K or PB2-L462F mutation. (A) Schematic diagram of the pUC57mini-D/OK-HEF-Reporter plasmid. This reporter plasmid contains the GFP reporter gene inserted between the D/OK-HEF cRNA 5′ and 3′ ends and then flanked by the Pol I terminator and the human RNA Pol I promoter. All ATG codons before the GFP translation initiation codon in the construct were mutated to CTGs; thus, the translation of GFP utilized its own start codon. (B) Virus titers for PB1-E697K and PB2-L462F mutants were determined by a TCID50 assay. Note that IDV OK-RGS660-PB1 is identical to IDV OK-RGS except for the PB1 segment derived from IDV D/660. (C to E) Activity of the IDV wild-type RNP complex or the RNP complex with the PB1-E697K or PB2-L462F mutation was measured by a minigenome replication assay. In this assay, the GFP reporter plasmid and the wild-type or mutant plasmids encoding IDV RNP complex components were cotransfected into HEK-293T cells. At 48 h posttransfection, cells were collected, and the GFP reporter was detected and analyzed by fluorescence microscopy (C), Western blotting (D), and FACS analysis (E). NP or β-actin was also detected by Western blotting, which was set up as a transfection or loading control, respectively. Note that densitometry of Western blot bands was quantified by using ImageJ software (https://imagej.nih.gov/ij/). Specifically, the density of the GFP band (i.e., surrogate of viral RNP activity) was first normalized by the value obtained with the input transfected NP control (i.e., to gauge the transfection variability). Relative viral RNP activity was determined by setting the level of the wild-type “OK-RNP” group to 1.00. (F) Mutations PB1-E697K and PB2-L462F are localized on the complex structure of RNA polymerase from ICV (PDB accession number 5D98). The structure of RNA polymerase was downloaded from the PDB (https://www.rcsb.org/) and was shown in PyMOL. The data presented in panels B to E are representative of results from three independent experiments, with each experiment analyzing samples in duplicate. eGFP, enhanced GFP.
- FIG 5
Activities of IDV and ICV RNP complexes with the conserved cognate or noncognate noncoding ends of vRNA segments. (A) Diagram showing nucleotide sequences of both the 3′ and 5′ noncoding regions in the M segment of influenza C and D viruses within the context of the GFP-based reporter. Note that the fifth nucleotide of the 3′ noncoding end is different between the M segment sequences of influenza C and D viruses and is in red with boldface type. “D(A→C)” indicates a mutated IDV reporter in which the fifth nucleotide, A, on the 3′ noncoding end of the D/OK M segment is altered to C, which makes it like the C/Vic M segment 3′ NCR in the first 11 nucleotides. (B to D) Activities of IDV and ICV RNP complexes with the cognate or noncognate promoter were determined by the minigenome replication assay. HEK-293T cells were cotransfected with the ICV or IDV M segment promoter sequence-based GFP reporter plasmid and the plasmids expressing RNP complex components from either D/OK or C/Vic. At 48 h posttransfection, cells were collected, and GFP reporter protein expression was analyzed by fluorescence microscopy (B), Western blotting (C), and FACS analysis (D). Renilla luciferase or β-actin detected by Western blotting was set up as a transfection or loading control. Note that densitometry of Western blot bands was quantified by using ImageJ software (https://imagej.nih.gov/ij/) (C). Specifically, the density of the GFP band (i.e., surrogate of viral RNP activity) was first normalized by the value obtained with the input transfected Renilla luciferase control (i.e., to gauge the transfection variability). Relative viral RNP activity was determined by setting the level of the wild-type group involving C-RNP/C-reporter or D-RNP/D-reporter (containing the respective cognate promoter) to 1.00. The data presented in panels B to D are representative of results from three independent experiments, with each experiment analyzing samples in duplicate.
Tables
- TABLE 1
Primers used in the RT-PCR and cloning experiments
