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. 2016 Feb;22(2):289-91.
doi: 10.3201/eid2202.151278.

Ebola Virus Persistence in Semen Ex Vivo

Ebola Virus Persistence in Semen Ex Vivo

Robert J Fischer et al. Emerg Infect Dis. 2016 Feb.

Abstract

On March 20, 2015, a case of Ebola virus disease was identified in Liberia that most likely was transmitted through sexual contact. We assessed the efficiency of detecting Ebola virus in semen samples by molecular diagnostics and the stability of Ebola virus in ex vivo semen under simulated tropical conditions.

Keywords: Ebola; Ebola diagnostics; Ebola virus disease; qRT-PCR; semen; sexual transmission; viruses.

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Figures

Figure 1
Figure 1
A) Standard curves of Ebola virus spiked into 3 matrices: semen, blood, and tissue culture medium. Samples were analyzed on the basis of 5 biologic replicates. PCR efficiency was from 98% in cell culture medium, 102% in semen, and 103% in blood. Analysis of covariance showed no significant difference (p<0.05) between the slopes of the linear regressions of blood and semen. B) Matrix dilution in which semen, blood, and tissue culture medium were 3-fold serially diluted in sterile physiologic saline solution and spiked with 104 TCID50/mL Ebola virus. The slopes for blood and medium did not differ significantly from 0. If the semen sample data are analyzed disregarding the undiluted sample, the resulting slope also does not differ significantly from 0. Ct, cycle threshold; TCID50, 50% tissue culture infectious dose. Error bars represent the mean ± SEM of 5 quantitative PCR analyses.
Figure 2
Figure 2
Linear regression model showing stability of Ebola virus (EBOV) and EBOV RNA in semen at 27°C and 80% relative humidity over 8 days. A) EBOV in bulk (liquid) semen versus dry semen at initial titers of 106 TCID50/mL and 103 TCID50/mL. The higher titer 1 × 106 TCID50/mL was used to provide a comparison with EBOV in blood, and the lower titer 1 × 103 TCID50/mL was derived from Ct values reported in semen samples. Viable virus was reduced significantly faster (p<0.0001) in dry semen than in bulk semen. The goodness-of-fit for the linear regression represented as the r2 value is 0.53 for bulk semen and 0.82 for dry semen with an initial titer of 106 TCID50/mL, respectively, and 0.65 for bulk semen with an initial titer of 103 TCID50/mL. No curve is shown for the initial titer 103 TCID50/mL in the dry semen because no viable virus was recovered after day 1. The titer on day 1 was 1.1 log10 TCID50/mL. In all cases except the high-titer bulk semen sample, the final data point was followed by 2 consecutive days of no recovered virus. B) Ct values produced by analysis of bulk semen samples analyzed by real-time quantitative reverse transcription PCR. The data did not fit a linear regression model (r2 = 0.08964), but the RNA clearly remained stable during the entire experiment. Three biologic replicates were analyzed at each time point. Error bars represent mean ± SEM virus titer. Dashed line indicates the limit of detection for the titration assay. An analysis of covariance was used to compare linear regression models and determine differences in virus reduction rates. Ct, cycle threshold; TCID50, 50% tissue culture infectious dose.

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