Potential Sexual Transmission of Zika Virus

Didier Musso; Claudine Roche; Emilie Robin; Tuxuan Nhan; Anita Teissier; Van-Mai Cao-Lormeau


Emerging Infectious Diseases. 2015;21(2):359-361. 

In This Article

The Study

In early December 2013, during the ZIKV outbreak, a 44-year-old man in Tahiti had symptoms of ZIKV infection: asthenia, low grade fever (temperature from 37.5°C to 38°C) and arthralgia. Symptoms lasted 3 days. Eight weeks later, he described a second episode of symptoms compatible with ZIKV infection: temperature from 37.5°C to 38°C, headache on days 1–3, and wrist arthralgia on days 5–7. The patient did not seek treatment, thus biological samples were not collected during the first 2 periods of illness. The patient fully recovered from the second episode, but 2 weeks later he noted signs of hematospermia and sought treatment. Because the patient had experienced symptoms of ZIKV infection some weeks before, he was referred to our laboratory in the Institut Louis Malardé, Papeete, Tahiti for ZIKV infection diagnostic testing. The medical questionnaire revealed no signs of urinary tract infection, prostatitis, urethritis, or cystitis, and the patient stated that he did not had any recent physical contact with persons who had acute ZIKV infection. We collected blood and semen samples. Direct and macroscopic examinations of the semen confirmed hematospermia. We extracted RNA using the NucliSENS easyMAG system (bioMérieux, Marcy ľEtoile, France) from 200 μL of blood and from 500 μL of semen and urine; both were eluted by 50 μL of elution buffer. We used 5 μL of RNA extracted for amplification. We tested blood and semen RNA extracts using real-time reverse transcription PCR (rRT-PCR) as described using 2 primers/probe amplification sets specific for ZIKV.[3] The rRT-PCR results were positive for ZIKV in semen and negative in blood, and confirmed by sequencing of the genomic position 858–1138 encompassing the prM/E protein coding regions of ZIKV. The generated sequence (GenBank accession no. KM014700) was identical to those previously reported at the beginning of the ZIKV outbreak.[3] Three days later, we collected a urine sample, then a second set of blood and semen samples. Semen and urine from this second collection were not found to contain traces of blood by both direct and macroscopic examinations. rRT-PCR detected ZIKV RNA in the semen and urine, but not in the blood sample.

We quantified ZIKV RNA loads using an RNA synthetic transcript standard that covers the region targeted by the 2 primers/probe sets. RNA loads were: 2.9 × 107 copies/mL and 1.1 × 107 copies/mL in the first and second semen samples, respectively, and 3.8 × 103 copies/mL in the urine sample.

We cultured semen and urine as described for dengue virus cultured from urine.[6] Briefly, 200 μL of each sample diluted in 200 μL of 1% fetal calf serum (FCS) minimum essential medium (MEM) were inoculated onto Vero cells and incubated for 1 h at 37°C; inoculum was then removed and replaced by 1 mL of culture medium. We also inoculated a negative control (200 μL of 1% FCS-MEM) and a positive control (5 μL of a ZIKV-positive serum diluted in 200 μL of 1% FCS-MEM). The cells were then incubated at 37°C in 5% CO2 for 6 days. The presence of ZIKV in the culture fluids was detected by rRT-PCR as described.

Replicative ZIKV particles were found in the 2 semen samples but none were detected in the urine sample. This finding does not exclude the possibility that ZIKV particles were present in urine. Positive samples were not titered.