Viruses characterized and associated stability values.

Viruses characterized and associated stability values.</p

<i>Renilla</i> luciferase assay of residual infectivity after acid exposure using MDCK Luc9.1 reporter cells.

MDCK Luc9.1 cells were inoculated with A/TN/09 WT (solid circles) or Y17H (open triangles) at MOI values ranging from 2x10-5 to 2 PFU/cell and were overlaid with media lacking TPCK-treated trypsin (A,B; -T) or containing it (C,D; +T). After 17 h, cell lysates were mixed with Renilla luciferase substrate and luminescence (relative light units, RLU) was measured using a 96-well plate luminometer. Virus aliquots in panels E-H were exposed to pH-adjusted PBS for 1h before inoculation into Luc9.1 reporter cells at an MOI of 2 (E,F) or 0.2 (G,H) PFU/cell. Data in panels E-H were fit by nonlinear regression (dose-response simulation) to calculate the midpoints of inactivation, or “inactivation pH”, which are listed on the panels. Dotted horizontal lines correspond to the lowest detectable residual infectivity after acid treatment (defined as 3 standard deviations above the mean of low-pH inactivated aliquots). Error bars represent standard deviation (n = 3). Reported data are representative of three independent experiments.</p

Comparison of percent virus inactivation and syncytia formation as a function of incubation pH.

The percent of virus inactivation by TCID50 assay was calculated at pH values ranging from 5.3 to 5.9 at 0.1-unit increments. These values are displayed to the right of associated micrographs. HA activation pH values by syncytia assay and inactivation pH values by luciferase assay are provided at the top of each column for reference. The viruses are A/TN/09 WT, A/swine/Wisconsin/11/1980 (H1N1), A/swine/California/T9001707/1991 (H1N1), and A/black-headed gull/Sweden/5/1999 (H16N2).</p

Virus inactivation measured by TCID50 and luciferase assays.

TCID50 values (left y-axis, black circles) and luciferase activity (RLU, right y-axis, white squares) were measured as a function of exposure to the reported pH followed by re-neutralization. Right y-axes are scaled so that RLU maxima and minima are displayed at levels similar to those from TCID50 on the opposite axis. Error bars represent standard deviation (n = 3). Reported data are representative of two or more independent experiments. (A) rg-A/TN/1-560/2009 (H1N1). (B) A/Bethesda/55/2015 (H3N2). (C) A/Aichi/2/1968 (H3N2). (D) rg-A/swine/NC/18161/2002 (H1N1). (E) A/swine/North Carolina/0033/2011 (H3N2). (F) A/swine/North Carolina/1256/2011 (H3N2). (G) A/swine/California/T9001707/1991 (H1N1). (H) A/canine/Illinois/41915/2015 (H3N2). (I) rg-A/canine/Indiana/1177-17-1/2017 (PR8 internal genes) (H3N2). (J) A/ruddy turnstone/NJ/65/1985 (H7N3). (K) A/duck/Australia/341/1983 (H15N8). (L) A/gull/Maryland/704/1977 (H13N6).</p

HA activation pH values measured by syncytia formation assay.

Viruses identified by the numbers on the left side of each row (cf. Table 1) were inoculated into Vero cells at an MOI of 3 PFU/cell. The pH values for PBS overlaid onto the cells are reported on the top left of each microscopic image. HA activation pH was defined as the highest pH at which syncytia formation occurred (third column).</p

Schematic of luciferase reporter assay to measure influenza virus inactivation pH.

(A) Acid treatment of samples. Into each well of a 96 deep-well plate, 495 μl of pH-adjusted PBS is added. 5-μl aliquots of twelve virus or control samples are added to eight wells each and incubated at 37°C for 1h. (B) Re-neutralization. A multichannel pipette is used to transfer 90 μl of sample from panel A into 810 μl infection media that has a pH of 7.0. (C) Infection of reporter cells. A multichannel pipette is used to transfer 200 μl re-neutralized virus to a white 96-well tissue culture (TC) dish containing MDCK-Luc9.1 cells. After 17-19h incubation at 37°C and 5% CO2, media is aspirated, 20 μl of lysis buffer is added, and 100 μl of diluted Renilla luciferase assay substrate is added. (D) Quantification of reporter gene expression and calculation of virus inactivation pH. Luminescence is measured using a plate-reader luminometer. Relative light units (RLUs) are output to csv files, which are then analyzed in GraphPad Prism 8 to calculate the point of inflection by nonlinear regression with a dose-response equation. In this example, samples include virus 1 (calculated pH 5.81), virus 2 (calculated pH 5.35), and virus 3 (control virus, used to estimate the baseline reading). RLU values were assigned for illustrative purposes only and are not real data. (E) Comparison of luciferase and TCID50 assays to measure virus inactivation pH. Total virus is an estimate of total PFUs needed for each assay. Time to conduct assay is after culturing of cells.</p

Virus thermostability of A/TN/09 variants and its relationship to pH stability.

Residual infectivity (normalized TCID50) was determined for virus-containing aliquots incubated at pH 7.0 for the reported times at 55°C (A), 37°C (D), and 33°C (G). Viruses are Y17H (yellow triangles), Y17H/V55I (orange triangles), WT (black circles), and V55I (blue squares). Virus numbers are 1–4. The 10% (dotted) line corresponds to a 90% reduction of starting infectivity. Virion inactivation pH and HA activation pH values are compared to calculated 90% Reduction time (Rt) values measured at 55°C (B-C), 37°C (E-F), and 33°C (H-I). Representative data from a repeated experiment is shown. Data were analyzed by simple linear regression using GraphPad Prism 8 and calculated R2 values are reported in the bottom-left corners.</p

Comparison of virus inactivation values measured by TCID₅₀ and luciferase assays.

Inactivation pH values measured byTCID50 are reported as the calculated pH corresponding to a 2-log10 (100-fold) drop in infectivity measured by TCID50 assay. Inactivation pH values for luciferase assay are reported as the pH at the midpoint of the S-shaped luminescence curves. Both TCID50 and luciferase inactivation values were calculated using GraphPad Prism 8 using a nonlinear regression with log(agonist) vs. response and a four-paramter variable slope. The displayed line (R2 value of 0.604) was calculated by simple linear regression using GraphPad Prism 8.</p

Virus thermostability of swine H1N1 and H3N2 isolates and its relationship to pH stability.

(A-D) Residual infectivity (normalized TCID50) determined for virus-containing aliquots incubated at pH 7.0 for the reported times at 55°C. Virus numbers and their associated symbols, HA activation pH, inactivation pH, and Rt values are listed below each panel. The 10% (dotted) line corresponds to a 90% reduction of starting infectivity. (E-F) Virion inactivation pH and HA activation pH values compared to calculated 90% Reduction time (Rt) values measured at 55°C. Representative data from a repeated experiment is shown. Data were analyzed by simple linear regression using GraphPad Prism 8 and calculated R2 values are reported in the top-right corners.</p

Acid inactivation titrations performed using 0.5 and 0.2 pH-unit steps.

Virus inactivation titrations were performed using Luc9.1 cells. Viruses used were A/TN/09 WT (solid circles), HA1-Y17H (open triangles), and HA2-R106K (gray squares) at an MOI 0.2 PFU/cell. Virus aliquots were incubated with pH-adjusted PBS at 0.5-unit steps (A-C) or 0.2-unit steps (D-F). After reneutralization in media supplemented with TPCK-treated trypsin, virus samples were loaded onto Luc9.1 cells and then incubated for 17 h before luminescence was measured as relative light units (RLU). Midpoints of inactivation, or inactivation pH values, are listed on the panels. Dotted lines correspond to the limit of detection (3 standard deviations above the mean) of uninfected negative control samples. Error bars represent standard deviation (n = 3). Reported data are representative of three independent experiments. (TIF)</p