Cell culture and virus preparation
Madin–Darby canine kidney (MDCK) cells (accession no. CCL-34, ATCC, Manassas, VA) were maintained in Dulbecco’s Modified Eagle’s Medium (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (Fujifilm Wako Pure Chemical Corporation), 100 U/mL penicillin, and 100 μg/mL streptomycin (Fujifilm Wako Pure Chemical Corporation) at 37 °C in a 5% CO2 atmosphere. IAVs (A/Panama/2007/1999 [H3N2] [Panama] and A/Puerto Rico/8/1934 [H1N1] [PR8]) were propagated in MDCK cells and the aliquots were stored as IAV stocks at ˗80 °C. The titer of the IAV stocks was determined using the 50% tissue culture infectious dose (TCID50) assay as described below.
Disinfection of IAV
Heat-inactivation was performed by heating the 140 μL IAV stocks at 100 °C for 1, 3, 5, 7.5, 10, and 15 min or 1, 3, and 5 min for Panama or PR8 strains, respectively, using a dry block incubator (MyBL-100, As One Corporation, Osaka, Japan) . After the heating, the heated IAV stocks were immediately put into an icebox to stop heating. The treated samples were used for the TCID50 assays or viral RNA isolation. The non-treated (0 min heated) samples (140 μL) were used for the TCID50 assays or viral RNA isolation without any special processing.
Sodium hypochlorite treatment was performed by incubating the mixtures of the 137.7 μL IAV stocks and 6% sodium hypochlorite (2.3 μL) for 10, 20, and 30 min or 3, 5, and 10 min for Panama or PR8 strains, respectively, according to published guidelines , then subsequently 10-fold diluted in phosphate-buffered saline (PBS) (−) (Fujifilm Wako Pure Chemical Corporation) to stop the treatment. The non-treated (0 min treated) samples (137.7 μL IAV stocks) were mixed with 2.3 μL PBS (−) and the mixtures were 10-fold diluted in PBS (−). The diluted samples were used for the TCID50 assays and viral RNA isolation.
For the ethanol treatment, the 42 μL IAV stocks were mixed with 100% ethanol (98 μL) and incubated for 10, 20, and 30 min , then 10-fold diluted in PBS (−) as performed in the sodium hypochlorite treatment. The non-treated (0 min treated) samples (42 μL IAV stocks) were mixed with 98 μL PBS (−) and the mixtures were 10-fold diluted in PBS (−). The diluted samples were used for the TCID50 assays and viral RNA isolation. The sodium hypochlorite treatment and ethanol treatment were performed at room temperature (approximately 20 °C) on a table. Regardless of the inactivation methods, three individual IAV suspensions per each time condition of inactivation were separately treated.
IAV infectious titer was analyzed by TCID50 assay according to the published studies with minor modifications [2, 5]. Briefly, MDCK cells were split into 96-well culture plates at a concentration of 2 × 104 cells/well. Regardless of the inactivation methods, a sample (non-treated or treated) was serially diluted 10-fold in the Eagle’s minimal essential medium (E-MEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 1 μg/mL trypsin (Sigma-Aldrich Japan, Tokyo, Japan). MDCK cells were inoculated with 100 μL of each IAV dilution and incubated at 37 °C for 1 h. The inoculum was removed, and the cells were washed once with PBS to eliminate the unbound virus particles. The cells were overlaid with the E-MEM and incubated at 37 °C for a week. The ratio of wells where cytopathic effect (CPE) was observed and calculated by multiplying the relevant dilution factors to calculate the infectious titer. Infectious titers were calculated using the Behrens–Karber method  and defined as the log of TCID50 units [log (TCID50/0.1 mL)]. The data represents the mean ± standard error of the three independent experiments. The limit of detection (LoD) of the TCID50 assays was 0.75 log (TCID50/0.1 mL). For convenience, the values of stock where no CPE [lower than 0.50 log (TCID50/0.1 mL)] was observed were considered as zero [log (TCID50/0.1 mL)] and were included as data points in the figures.
PA was analyzed using LR-RT-qPCR to evaluate the effects of inactivation treatments in this study . Regardless of the inactivation methods, viral RNA was isolated from 140 μL sample (non-treated or treated) using the QIAamp Viral RNA Mini Kit (Qiagen K.K., Tokyo, Japan) according to the manufacturer’s instructions. Each RNA sample was eluted in 60 μL of elution buffer. The isolated RNA was subjected to reverse-transcription using SuperScript III (Thermo Fisher Scientific K.K., Tokyo, Japan) and an RT primer (5′-AGCGAAAGCAGG-3′) that binds specifically at the 3′ terminus of IAV PA . The isolated RNA was then digested using RNase H included in SuperScript III. qPCR was then performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific K.K.) according to the manufacturer’s instructions. Thermal cycling was performed on the LightCycler 96 (Roche Diagnostics K.K., Tokyo, Japan) under the following cycling conditions: activation at 95 °C for 10 min, and 40 cycles of PCR at 95 °C for 15 s and 60 °C for 1 min. The primer pair for the qPCR was Fw: 5′-GGATTTTCAGCGGAGTCAAG-3′ and Rev.: 5′-GGAGTTGAACCAAGACGCAT-3′ as used in the previous study . DNA standard samples, measured the concentration by a spectrophotometer Nanodrop (Thermo Fisher Scientific K.K., Tokyo, Japan), were 10-fold serially diluted and subjected to the RT-qPCR analyses at the same time with the IAV samples to make standard curves. The IAV standard curve was created by measuring the Ct values of a serially diluted plasmid DNA containing the PCR amplicon . Values below that of the most diluted standard were defined as “not detected” and included in the datasets as zero. Therefore, the LoD (cut-off copy number) of the LR-RT-qPCR assay for the PA segment was 5.53 copies/μL. Copy number quantification was performed using the IAV standard curves. Each copy number was divided by the copy number of non-treated IAV to calculate the copy number ratio. Data represent the three independent experiments and the mean ± standard error of them.
The significance of all the datasets in the TCID50 and LR-RT-qPCR assay was verified using the Kruskal–Wallis test. The Dunn–Bonferroni post hoc method was conducted to assess the significance between each pair of datasets of averaged technical replicates in the TCID50 and LR-RT-qPCR assay. Values considered to be significant had P-values < 0.05.