Abstract
We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.
Highlights
Zika virus (ZIKV) was first identified in 1947 in Africa and subsequently reached Asia and, more recently, the Americas [1,2]
BEAST v1.8.3 [23] was used to estimate dN/dS and the rate of ZIKV evolution within the male reproductive system (MRS), HyPhy v2.3 was used to identify codons with evidence for positive, diversifying selection, and for samples with >50× average coverage, we examined intra host genetic variation using FreeBayes v1.0.2 [24]
ZIKV01 was the only patient in this study that developed viremia, which was detected at day 5 (6.2 × 107 genome equivalents/mL) and lasted until day 12 (2.2 × 102 ge/mL)
Summary
Zika virus (ZIKV) was first identified in 1947 in Africa and subsequently reached Asia and, more recently, the Americas [1,2]. More than 80 countries around the globe have reported cases of active ZIKV transmission and the recent outbreaks have associated the virus with several neurological disorders, including Guillain-Barré syndrome and congenital neurologic birth defects [3,4,5,6]. Reports have shown that ZIKV RNA is present in several body fluids, including urine, blood, saliva, breast milk and secretions from the vaginal tract [7,8,9,10], being cleared early from serum but still detected in whole blood, saliva and urine for more than two weeks after symptoms onset [11,12,13,14]. The potential for persistent ZIKV infections in the MRS raises concerns ranging from the demonstrated sexual transmission of the virus to the damage of germ cells and generation of poor-quality sperm
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