Detection of viral RNA in tissues following plasma clearance from an Ebola virus infected patient

Abstract

Purpose: An unprecedented Ebola outbreak occurred in 2014-2015 in West Africa. A better understanding of the EBOV life cycle is fundamental to develop new countermeasures, as well as to fully comprehend the pathways of inter-human transmission. We have explored the possibility of viral persistence in different body fluids’ samples obtained from a Health Care Worker (HCW) infected in Sierra Leone and treated at INMI L. Spallanzani, Italy. To evaluate whether the virus was in a replicative status or simply derived from blood spill over, we compared the trends of EBOV-specific negative sense genomic RNA (neg-RNA), positive sense RNA (pos-RNA) and total viral RNA in different clinical samples. Methods & Materials: Clinical samples were inactivated in BSL4 facility and RNA was extracted with QIAamp Viral-RNA Mini-Kit (Qiagen). Quantification of total viral RNA was performed with the reference test Altona-FilovirusScreen Kit1.0 (Altona Diagnostics). To measure EBOV-specific negative sense genomic RNA (neg-RNA) and positive sense RNA (pos-RNA) [including both replication intermediate (cRNA) and messenger RNA (mRNA)], L-gene specific reverse or forward primers were used in the reverse transcription step Results: The clinical samples analyzed (sputum, nasopharyngeal swab, ocular swab, urine and plasma) show different trends in neg-RNA, pos-RNA and total viral RNA levels. In Nasopharyngeal swab, Ocular Swab and Urine, total viral RNA is the only one detectable, until Day 12, 5 and 15 after hospitalization, respectively. On the contrary, in plasma, total viral RNA, neg-RNA and pos-RNA levels simultaneously decreased, starting from day 3 and becoming undetectable at Day 6. In Sputum, pos-RNA levels decreased since Day 8, persisting at detectable levels up to Day 10, coherently with total viral RNA and neg-RNA levels, which start decreasing at Day 10 and become undetectable at Day 11. Conclusion: The presence of viral RNA and replication markers (pos-RNA and neg-RNA) in sputum might be of relevance in future analysis regarding the persistence of the virus in the upper respiratory tract, even after viral clearance from plasma. These results should be taken under further investigation in order to better understand the role of the respiratory tract for possible involvement in viral shedding, viral replication site or as a viral reservoir.