RNA secondary structure analysis
Influenza virus NS gene nucleotide sequences were downloaded from the NCBI Influenza Virus Resource  and Influenza Research Database (IRD) . RNA secondary structures were predicted using the RNAfold online tool , which does not include pseudoknot prediction.
The pHW-PR8-NS plasmid, encoding the NS segment of the A/PR/8/34 (H1N1) virus, was used as a starting point. PCR with primers featuring incompletely overlapping sequences was used to introduce mutations, as described by Zheng et al. . The primers used for PCR site-directed mutagenesis are listed (Additional file 1: Table S1). Mutations were first introduced into the 82–148 region, then into the 497–564 region. Four plasmid variants were obtained, and modifications were verified by Sanger DNA sequencing .
In vitro transcription
RNA fragments of NS regions 82–148 and 496–564 were prepared by in vitro transcription. At first, PCR products containing the regions of interest were amplified from modified pHW-PR8-NS plasmids, and T7 promoter sequences were added via the primers used (Additional file 2: Table S2). Next, RNA regions were synthesized by T7 RNA Polymerase (Promega, #P207B) according to the enzyme manufacturer’s instructions. Following RNA synthesis, template DNA was removed by subsequent digestion with DNase (Promega, #M6101). Transcripts were purified by double isopropanol precipitation .
Electrophoretic mobility under native and denaturing conditions
For analysis under denaturing conditions, 1 μg of each RNA sample was mixed with an equal volume of 2x RNA Gel Loading Dye (Thermo Scientific, #R0641), incubated for 30 min at room temperature, and then heated for 3 min at 95 °C. Samples were subsequently loaded onto a PAGE (12.5% polyacrylamide/8 M urea) and run in TBE buffer at 55 °C. For analysis under native conditions, samples were incubated for 2 min at 90 °C, followed by slow cooling (approx. 2 degrees per 20 s) until 37 °C, and further incubated for 25 min at 37 °C, as described by the Moss group . Samples were then mixed with 6x gel loading buffer without formamide (0.25% bromophenol blue (w/v), 0.25% xylene cyanol FF (w/v), 40% sucrose (w/v)), loaded onto a 12.5% polyacrylamide gel, and run in 0.5xTBE buffer at 37 °C. Gels were stained with silver nitrate , and images were captured using a ChemiDoc XRS+ System (Bio-Rad).
MDCK London line cells (International Reagent Resource, #FR-58) were cultivated in Alpha MEM (Biolot) supplemented with 2 mM L-glutamine and 10% “SC-biol” FBS (Biolot). A549 cells were cultivated in DMEM/F12 (Gibco) media supplemented with 2% GlutaMAX (Gibco) and 10% FBS (Gibco). Vero cells (ATCC, #CCL-81) were adapted to serum-free medium and cultivated in OptiPro SFM (Gibco) supplemented with 2% GlutaMAX (Gibco).
Generation of viruses
Viruses were obtained by reverse genetics . Plasmids encoding 8 gene segments of A/PR/8/34 (H1N1) including mutated NS segments were transfected into Vero cells by Nucleofector technology (Amaxa #VCA-1003). Viruses were collected from the supernatant 72 h post transfection, and working stock was generated by one passage in Vero cells at moi = 0.01. Virus infectious activity was measured by titration in Vero cells. The 50% Tissue Culture Infectious Dose (TCID50) endpoint was calculated by the Reed and Muench method . Virus genome sequences, including the mutations introduced, were confirmed by next generation sequencing (NGS).Full-genome amplification was performed according to Zhou et al. ). Nextera XT (Illumina) sample preparation was used to obtain libraries for NGS; full-length genome sequences were obtained using Illumina MiSeq.
Viral growth kinetics
Multi-cycle viral growth kinetics were measured in Vero, MDCK, and A549 cells (overnight confluent, 6-well plate format, triplicates) by infection with virus at moi = 0.001 (TCID50/cell). After 1 h of virus adsorption at room temperature, medium was replaced. Serum free culture media (see above) was supplemented with 1% antibiotic-antimycotic (Gibco) and TPCK-trypsin (Sigma) at: 2.5 μg/ml for MDCK; and 0.5 μg/ml for A549 and Vero. Viral progeny was collected from the supernatant at the indicated time points, and infectious titers were measured.
MDCK or A549 monolayers (overnight confluent, 48-well plate format, triplicates) were infected with assembled viruses at moi = 10. After virus adsorption at 37 °C, the inoculum was removed and replaced with serum free medium containing antibiotic-antimycotic and TPCK-trypsin. Infected cells were incubated at 37 °C for the indicated time periods and then fixed with 80% acetone in DPBS (ice cold at application) for 15 min at room temperature. Fixed cells were washed three times with phosphate buffered saline with 0.1% tween-20 (PBST) and blocked with 5% milk in PBST for 6 h at 4 °C. After 2 washes, plates were incubated at 4 °C overnight with mouse polyclonal serum raised against recombinant NS1(1–124) protein. Plates were then washed three times with PBST, and goat anti-mouse secondary antibody (Bio-Rad) was added (1 μg/ml final), followed by incubation at 37 °C for 1 h. Immunoreactivities were analyzed by adding TMB Peroxidase EIA Substrate (Bio-Rad), and reactions were stopped with 2 N H2SO4. Optical density was measured at 450 nm with removal of noise (measured at 655 nm) using a CLARIOstar multi-function reader (BMG Labtech).
Real-time PCR analysis
MDCK or A549 cell monolayers (overnight confluent, 6-well plate format, triplicates) were used for infection at moi = 1. After virus adsorption at 37 °C, the inoculum was removed and replaced with serum free medium. At the indicated time points, media was removed, and total RNAs were extracted from cells using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Complementary DNA was synthesized from 500 ng of total RNA using M-MLV Reverse Transcriptase (Promega) and oligo(dT16) primers (DNA synthesis, Russia) according to the manufacturer’s protocol. Quantitative real-time PCR analysis was performed using (2x) BioMaster HS-qPCR SYBR Blue (Biolabmix). The primers used are provided (Additional file 3: Table S3). NS1 gene expression was calculated by the 2−ΔΔCtmethod, and GAPDH mRNA was used for normalization. The average value of the “reference sample” replicates (“1–1” sample at 1 h post-infection, h.p.i.) was designated as expression level ‘1.0’ and used for relative expression quantitation.
Statistical analysis was performed using GraphPad Prizm 6.01 software. One-way ANOVA was performed to evaluate differences in virus infectious activity. Two-way ANOVA with Tukey post test was used to evaluate the significance of any differences between viruses at specific time points, revealed by ELISA and RT-PCR. A value of 0.05 was used as the threshold of significance. All samples were processed in triplicate, and presented on graphs by mean ± sd.