Evidence of Ebola Virus Replication and High Concentration in Semen of a Patient During Recovery

Abstract

In one patient over time, we found that concentration of Ebola virus RNA in semen during recovery is remarkably higher than blood at peak illness. Virus in semen is replication-competent with no change in viral genome over time. Presence of sense RNA suggests replication in cells present in semen.

Keywords: Ebola; persistence; replication; semen; sexual transmission.

Figure 1.

High viral load and identical genomes within a single patient with Ebola virus disease. A, Viral load in semen and serum. We measured viral load from extracted RNA using primers in Supplementary Table 2 (Trombley et al, 2010). Samples were quantified against a standard curve (assay limit of detection [LOD]: 1 copy/µL) to determine copies per microliter of extracted RNA. Based on the different extraction methods, we converted these values into copies per milliliter of indicated fluid (blue: serum, n = 6 replicates per sample; orange: semen, n = 3). Asterisk indicates below-assay LOD. B, Quantification of Ebola virus (EBOV) antisense (vRNA) and sense (c/mRNA) RNA in semen samples using strand-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). We designed RT primers for a 2-step RT-qPCR to detect antisense or sense viral RNA separately. Day 180 semen sample was not tested. C, Quantification of EBOV vRNA and c/mRNA in semen samples using single-molecule tagged amplicon sequencing. We uniquely tagged and amplified RNA molecules with random barcodes in a strand-specific manner. We deduplicated reads and counted unique numbers of barcodes. Molecules at day 180 likely represent background amplification. See Supplementary Figure 1C for quantification in peripheral blood leukocyte (PBL) samples. D, Deep sequencing metrics. We performed RNA-seq either without (unbiased) or with hybrid selection using EBOV-specific baits to reduce host RNA background. Lower viral genome coverage is observed in PBL and semen samples due to abundant cellular RNA. E, Phylogeny of the SL4 clade. We combined 1489 publicly available genomes (Diehl et al 2016) with this patient’s viral genome and generated a phylogenetic tree with 1000 bootstrap replicates. The patient sample falls within a SL4 subclade (maroon; 100% bootstrap support) of samples collected near Freetown, Sierra Leone, from late January 2015 through March 2015, representing a likely transmission network. See Supplementary Figure 2 for full tree.