Differences in the Comparative Stability of Ebola Virus Makona-C05 and Yambuku-Mayinga in Blood.

In support of the response to the 2013-2016 Ebola virus disease (EVD) outbreak in Western Africa, we investigated the persistence of Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 (EBOV/Mak-C05) on non-porous surfaces that are representative of hospitals, airplanes, and personal protective equipment. We performed persistence studies in three clinically-relevant human fluid matrices (blood, simulated vomit, and feces), and at environments representative of in-flight airline passenger cabins, environmentally-controlled hospital rooms, and open-air Ebola treatment centers in Western Africa. We also compared the surface stability of EBOV/Mak-C05 to that of the prototype Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga (EBOV/Yam-May), in a subset of these conditions. We show that on inert, non-porous surfaces, EBOV decay rates are matrix- and environment-dependent. Among the clinically-relevant matrices tested, EBOV persisted longest in dried human blood, had limited viability in dried simulated vomit, and did not persist in feces. EBOV/Mak-C05 and EBOV/Yam-May decay rates in dried matrices were not significantly different. However, during the drying process in human blood, EBOV/Yam-May showed significantly greater loss in viability than EBOV/Mak-C05 under environmental conditions relevant to the outbreak region, and to a lesser extent in conditions relevant to an environmentally-controlled hospital room. This factor may contribute to increased communicability of EBOV/Mak-C05 when surfaces contaminated with dried human blood are the vector and may partially explain the magnitude of the most recent outbreak, compared to prior outbreaks. These EBOV persistence data will improve public health efforts by informing risk assessments, structure remediation decisions, and response procedures for future EVD outbreaks

Two-Center Evaluation of Disinfectant Efficacy against Ebola Virus in Clinical and Laboratory Matrices

Ebola virus (EBOV) in body fluids poses risk for virus transmission. However, there are limited experimental data for such matrices on the disinfectant efficacy against EBOV. We evaluated the effectiveness of disinfectants against EBOV in blood on surfaces. Only 5% peracetic acid consistently reduced EBOV titers in dried blood to the assay limit of quantification

Comparison of EBOV/Mak-C05 Recovery by qRT-PCR and Viability by Microtitration Assay.

<p>The virus samples recovered from test surface coupons from Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148476#pone.0148476.g002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148476#pone.0148476.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148476#pone.0148476.g004" target="_blank">4</a> were analyzed by both qRT-PCR and virus microtitration assay to determine whether virus material was efficiently recovered at low viability timepoints (n = 3). qRT-PCR analysis was performed on samples aged at (A) 22°C/41% RH, or (B) 28°C/90% RH. The ratio of virus genomic copies recovered from timepoints at the microtitration limit of detection to recovery from timepoints immediately post-drying was determined and plotted as a percentage (black bars). This was compared to the virus viability ratio from the same timepoints, as determined by microtitration assay (Log TCID₅₀/mL) (gray bars). Actual mean percent recovery values are shown above each bar. Because no viable virus was detected at any timepoint in feces and % viable virus could not be calculated, % viable virus recovered in this matrix is indicated at <1%.</p

EBOV/Mak-C05 Surface Persistence in Simulated Vomit at Three Different Environments.

<p>The surface-specific decrease in EBOV/Mak-C05 viability in simulated vomit was measured at (A) 22°C/17% RH (stainless steel and polypropylene only), (B) 22°C/41% RH, or (C) 28°C/90% RH. The microtitration assay limit of detection (0.7 Log TCID₅₀/mL) is indicated by a gray bar at the bottom of each graph.</p

EBOV Surface Persistence Study Design.

<p>Vero E6-cultured stocks of EBOV/Mak-C05 were diluted 1:10 into the clinical test matrices. Ten μL drops (containing 10^6.4 TCID₅₀/mL) of each virus/matrix mixture were deposited onto sterile test surface coupons, dried 1–4 hours (environment dependent) in an environmentally-controlled chamber, and then aged in the same chamber at the indicated environment for 3–10 days. Approximately 10^4.4 (~25,000) TCID₅₀ were deposited on each test surface coupon. Viable virus was recovered from surface coupons immediately after deposition (wet), immediately after drying, and at a minimum of three subsequent timepoints. At each timepoint, triplicate samples were analyzed for virus viability by microtitration assay. Four hospital and PPE-associated surfaces were tested against multiple environments. For comparison, a subset of surfaces and environmental conditions (indicated by dotted box, bolded font, and an *) were used to determine the comparative persistence of EBOV/Yam-May.</p

EBOV/Mak-C05 Surface Persistence in Cell Culture Media at Three Different Environments.

<p>The surface-specific decrease in EBOV/Mak-C05 viability in cell culture media was measured at (A) 22°C/17% RH (stainless steel and polypropylene only), (B) 22°C/41% RH, or (C-D) 28°C/90% RH. The microtitration assay limit of detection (0.7 Log TCID₅₀/mL) is indicated by a gray bar at the bottom of each graph. The apparent increase at the 72 h timepoint in panel A is most likely due to experimental variation in replicate samples and not an increase in viral titer. The graphs in panels C-D are derived from two separate studies, as described in the methods section.</p

EBOV Viability Decay Rates and Half-lives for Each Test Matrix and Environment.

<p>A least-squares method was used to calculate the slope of the line defined by the post-drying surface-averaged points for each matrix and environment. This slope was used to calculate daily decay rate means (reduction in EBOV LogTCID₅₀/day) and 95% confidence interval (CI) for EBOV/Mak-C05 and EBOV/Yam-May. Mean half-lives (in hours) and ranges for each matrix and environment are indicated in bold. ND = not determined. NP = no persistence.</p

Comparative EBOV Viability Decay Rates and Stability in Dried Human Whole Blood.

<p>(A-B) Surface-composite decay plots in dried blood for EBOV/Mak-C05 and EBOV/Yam-May at 22°C/41% RH (A), and 28°C/90% RH (B). Viability values from stainless steel, TyChem QC, polypropylene (for both EBOV variants), and nitrile (for EBOV/Mak-C05 only) surfaces were combined at each timepoint (a minimum of n = 9 for each timepoint) and the mean viability was plotted v. post-drying time, using a least squares analysis, to derive surface-independent decay rates for each virus variant. These rates were used to calculate mean virus viability half-lives (in hours) at the two different environments in dried blood, which are indicated next to each decay line. Pre-drying timepoints are not included. Virus variants are indicated in the legend in panel A (EBOV/Mak-C05, blue dots, EBOV/Yam-May, red triangles).</p

Drying in Blood Reduces Viability of EBOV/Yam-May More Than EBOV/Mak-C05.

<p>Drops of human whole blood (10 μL) spiked with either EBOV/Mak-C05 (blue) or EBOV/Yam-May (red) were spotted onto surface coupons. The spots were either recovered immediately (pre-dry) or dried at (A) 22°C/41% RH for one hour, or (B) 28°C/90% RH for four hours, and then recovered (post-dry). Viable virus titers in each recovered sample were determined, and the difference between pre- and post-dry samples were compared. The T = 0 timepoints (n = 3) for each graph represent pre-drying virus viability values, and the second point (one hour post-deposition at 22°C/41% RH and four hours post-deposition at 28°C/90% RH) shows virus viability immediately post-drying (highlighted in gray). Virus variants are indicated in the legend in panel A (EBOV/Mak-C05, blue dots, EBOV/Yam-May, red triangles). (C) The difference in the reduction in viable EBOV during drying in cell culture media, whole blood, and simulated vomit is shown.</p

Environment-Dependent EBOV Viability Decay and Half-lives in Dried Human Whole Blood.

<p>(A-C) The surface-specific decrease in EBOV/Mak-C05 viability in dried blood was measured at (A) 22°C/17% RH for 72 h, (B) 22°C/41% RH for 96 h, or (C) 28°C/90% RH for 240 h. (D) Because ANOVA indicated that surface type did not significantly affect virus persistence, EBOV/Mak-C05 viability values from all surfaces were combined for each timepoint (a minimum of n = 6 for each timepoint) and the mean surface viability was plotted v. post-deposition time. A least-squares analysis was used to plot surface-independent virus viability decay at each environment (∎ 28°C/90% RH, solid line, ▲ 22°C/41% RH, dashed line, ◆22°C/17% RH, dotted line) and to derive surface-independent decay rates. These rates were used to calculate mean virus viability half-lives and 95% confidence intervals at each environment, and can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148476#pone.0148476.t001" target="_blank">Table 1</a>. The microtitration assay limit of detection (0.7 Log TCID₅₀/mL) is indicated by a gray bar at the bottom of panels A-D.</p