abstract n engl j med 378;15 nejm.org April 12,

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1 The new england journal of medicine established in 1812 April 12, 2018 vol. 378 no. 15 Zika Virus Shedding in Semen of Symptomatic Infected Men Paul S. Mead, M.D., M.P.H., Nisha K. Duggal, Ph.D., Sarah A. Hook, M.A., Mark Delorey, Ph.D., Marc Fischer, M.D., M.P.H., Dana Olzenak McGuire, Ph.D., Heidi Becksted, M.P.H., Ryan J. Max, M.P.H., Michael Anishchenko, Ph.D., Amy M. Schwartz, M.P.H., Wen Pin Tzeng, Ph.D., Christina A. Nelson, M.D., M.P.H., Erin M. McDonald, Ph.D., John T. Brooks, M.D., M.P.H., Aaron C. Brault, Ph.D., and Alison F. Hinckley, Ph.D. abstract BACKGROUND Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has been linked to adverse birth outcomes. Previous reports have shown that person-to-person transmission can occur by means of sexual contact. METHODS We conducted a prospective study involving men with symptomatic ZIKV infection to determine the frequency and duration of ZIKV shedding in semen and urine and to identify risk factors for prolonged shedding in these fluids. Specimens were obtained twice per month for 6 months after illness onset and were tested by realtime reverse-transcriptase polymerase-chain-reaction (RT-PCR) assay for ZIKV RNA and by Vero cell culture and plaque assay for infectious ZIKV. RESULTS A total of 1327 semen samples from 184 men and 1038 urine samples from 183 men were obtained 14 to 304 days after illness onset. ZIKV RNA was detected in the urine of 7 men (4%) and in the semen of 60 (33%), including in semen samples from 22 of 36 men (61%) who were tested within 30 days after illness onset. ZIKV RNA shedding in semen decreased substantially during the 3 months after illness onset but continued for 281 days in 1 man (1%). Factors that were independently associated with prolonged RNA shedding included older age, less frequent ejaculation, and the presence of certain symptoms at the time of initial illness. Infectious ZIKV was isolated from 3 of 78 semen samples with detectable ZIKV RNA, all obtained within 30 days after illness onset and all with at least 7.0 log 10 ZIKV RNA copies per milliliter of semen. From the Centers for Disease Control and Prevention (CDC) National Center for Emerging and Zoonotic Infectious Diseases, Fort Collins, CO (P.S.M., N.K.D., S.A.H., M.D., M.F., H.B., R.J.M., M.A., A.M.S., W.-P.T., C.A.N., E.M.M., A.C.B., A.F.H.); and the CDC National Center for Chronic Disease Prevention and Health Promotion (D.O.M.) and the CDC National Center for HIV AIDS, Viral Hepatitis, STD, and TB Prevention (J.T.B.) both in Atlanta. Address reprint requests to Dr. Mead at the Division of Vector- Borne Diseases, Centers for Disease Control and Prevention, 3156 Rampart Rd., Fort Collins, CO 80521, or at pmead@ cdc.gov. Drs. Mead and Duggal contributed equally to this article. N Engl J Med 2018;378: DOI: /NEJMoa Copyright 2018 Massachusetts Medical Society. CONCLUSIONS ZIKV RNA was commonly present in the semen of men with symptomatic ZIKV infection and persisted in some men for more than 6 months. In contrast, shedding of infectious ZIKV appeared to be much less common and was limited to the first few weeks after illness onset. (Funded by the Centers for Disease Control and Prevention.) n engl j med 378;15 nejm.org April 12,

2 The new england journal of medicine Zika virus (ZIKV) is a single-stranded RNA virus that is transmitted by aedes mosquitoes. The illness in humans is generally mild. Nevertheless, infection has been linked to congenital microcephaly and other adverse pregnancy outcomes and to the Guillain Barré syndrome. 1,2 Originally discovered in Uganda, ZIKV has emerged recently in the Pacific region and in the Americas, where it has spread rapidly to more than 48 countries, including the United States. 3,4 Sexual transmission of ZIKV was first posited in 2011 by a researcher whose wife became ill after his return from Senegal to the United States. 5 The virus was subsequently detected in the semen of a Tahitian man in whom hematospermia had developed after an acute febrile illness. 6 With the emergence of ZIKV in the Americas, confirmed or probable cases of sexual transmission have now been reported in at least 13 countries Most documented cases have involved sexual transmission from symptomatic men to women, although transmission from a man to another man, from a woman to a man, and from an asymptomatic man to a woman has been reported The potential implications of sexual transmission of ZIKV are far-reaching Perhaps most importantly, it has been proposed that sexual transmission might pose a risk of fetal infection that is greater than the risk associated with mosquito-borne transmission. 18 If so, the interruption of sexual transmission could play a critical role in preventing the serious complications that have been associated with fetal infection. Public health agencies in several countries have issued interim guidance recommending that condomless sexual contact with men who have been exposed to ZIKV or who have ZIKV infection should be avoided especially by women who are or may become pregnant for periods ranging from 8 weeks to at least 6 months after symptom onset or the last potential ZIKV exposure. 13,21,22 In subsequent reports, ZIKV RNA was found in the semen of infected men more than 180 days after illness onset, arousing concern that the period of avoidance might need to be lengthened. 23,24 To better inform public health recommendations, we conducted a prospective study involving 225 men with confirmed, symptomatic ZIKV infection. Our goals were to determine more precisely the frequency, duration, and pattern of ZIKV shedding in semen and urine and to identify risk factors for prolonged shedding in these body fluids. Methods Enrollment and Specimens Cases of ZIKV infection were identified by state and local health departments on the basis of reports from clinicians and laboratories. Cases were confirmed according to the laboratory criteria listed in the 2016 Council of State and Territorial Epidemiologists interim surveillance case definition. 25 Local health authorities contacted men who had confirmed symptomatic infection, informed them of the study, and referred interested candidates to staff of the Centers for Disease Control and Prevention (CDC) for screening, obtaining of informed consent, and enrollment. Enrollment was limited to men 18 years of age or older who resided in the continental United States or Hawaii. Men were excluded if they were incarcerated or did not speak English or Spanish. Baseline information was obtained, and a collection kit with return postage was mailed to the participant s home. Participants were asked to provide urine and semen samples, obtained in that order. Specimens were shipped overnight with cold packs. Additional kits were sent every 2 weeks thereafter, until 6 months from the date of the participant s onset of illness. Participants whose specimens still tested positive for ZIKV RNA at the end of 5 months were allowed to continue submitting samples until two consecutive samples tested negative. With each specimen submission, participants were asked to complete a seven-question survey regarding current urogenital symptoms and frequency of ejaculations in the 7 days before the sample was obtained. Testing results were provided to the enrolled participants at the end of participation. The study was approved by the CDC institutional review board and by the Office of Management and Budget, and oral informed consent was obtained from all the participants. Laboratory Testing Semen and urine samples were tested at the CDC Division of Vector-Borne Diseases. ZIKV RNA was assayed by real-time reverse-transcriptase polymerase-chain-reaction (RT-PCR) assay with 1378 n engl j med 378;15 nejm.org April 12, 2018

3 Zika Virus Shedding in Semen the use of Asian genotype specific primers, with a lower limit of detection of 4 RNA copies per reaction or 800 RNA copies per milliliter (for details of the assays and validation, see the Supplementary Appendix, available with the full text of this article at NEJM.org). Semen samples with ZIKV RNA that was detectable by RT-PCR were tested at a 1:10 dilution by Vero cell plaque assay for infectious particles. 26 Samples were selected for culturing nonrandomly, with preference given to samples that had been obtained sooner after illness onset and samples with higher viral RNA loads. The 1:10 dilution was necessary to prevent nonspecific cytopathic effects that may occur when undiluted semen samples with undetectable levels of viral RNA are inoculated onto Vero cells. Viral isolation was also attempted by inoculating diluted semen samples onto confluent monolayers of Vero cells in individual wells of six-well plates; viral replication was verified by using RT-PCR to test supernatant at multiple time points after inoculation for increasing levels of viral RNA. 27 Statistical Analysis Risk factors for viral RNA shedding were assessed by fitting a Weibull distribution to the number of days between illness onset and the clearance of ZIKV (see the Supplementary Appendix). Factors that were evaluated for association with duration of RNA shedding were the age of the participant, vasectomy history, average number of ejaculations per week during the study (not counting when the samples were obtained), ongoing pain or burning with urination or blood in the semen, and symptoms at the time of the initial illness, specifically the presence of fever, joint pain, rash, or conjunctivitis. Interaction was assessed for each possible pairing of risk factors. Results Study Population Among the 274 eligible men who were contacted, 225 were enrolled, and 185 participated by submitting at least one semen sample or one urine sample (Fig. S1 in the Supplementary Appendix). There were no significant differences in terms of age or symptoms of illness between enrollees who submitted specimens and those who did not submit specimens. Participants were residents of 24 U.S. states, the median age of the participants Table 1. Characteristics of the 185 Enrolled Participants Who Submitted Samples. Characteristic Value Symptom of initial illness no./total no. (%) Rash 176/183 (96) Fever 138/174 (79) Joint pain 140/183 (77) Conjunctivitis 95/177 (54) Bloody semen 8/177 (5) History of vasectomy no./total no. (%) 29/176 (16) Time from illness onset to first sample no. (%) days 40 (22) days 70 (38) days 28 (15) 91 days 47 (25) was 37 years (range, 18 to 69), and all but 1 participant had become infected during international travel. The laboratory basis for diagnosis was the detection of ZIKV RNA in a body fluid by RT-PCR in 183 of 225 men (81%), with or without serologic testing, and by serologic testing in 40 men (18%). A total of 2 men (1%) received a diagnosis of confirmed ZIKV infection as a result of testing of an unknown type. The majority of participants reported rash, fever, joint pain, and conjunctivitis as features of their initial illness, and 8 of 177 (5%) reported seeing blood in their semen during or shortly after illness onset (Table 1). A total of 2 men (1%) had disease that was linked to sexual transmission, and 29 of 176 men (16%) reported having undergone vasectomy. ZIKV RNA in Semen A total of 1327 semen samples were submitted by 184 participants, with a range of 1 to 19 samples per participant (median, 4 samples); 82% of the samples were received within 1 day after they had been obtained by the participant. The median time from illness onset to collection of the participant s first sample was 42 days (range, 14 to 222) (Table 1); the last sample for any participant was obtained 304 days after illness onset. Overall, participants reported ejaculating an average of 3 times (range, 0 to >10) during the 7 days before the sample was obtained; less than 3% of the participants reported dysuria or other genitourinary symptoms. n engl j med 378;15 nejm.org April 12,

4 The new england journal of medicine Table 2. Zika Virus (ZIKV) RNA Detection in Semen and Urine Samples over Time, According to Sample and Participant. Time since Illness Onset Positive Result in Semen Positive Result in Urine Samples Participants Samples Participants no./total no. (%) days 22/40 (55) 22/36 (61) 5/42 (12) 5/40 (12) days 76/172 (44) 48/112 (43) 3/172 (2) 3/107 (3) days 42/217 (19) 28/131 (21) 0/217 0/ days 13/229 (6) 9/132 (7) 0/225 0/ days 10/229 (4) 8/137 (6) 0/200 0/ days 6/247 (2) 2/141 (1) 0/145 0/91 >180 days* 5/96 (5) 1/91 (1) 0/37 0/36 Total 174/1230 (14) 60/184 (33) 8/1038 7/183 (4) * Data include those collected during extended sampling in men whose samples remained positive for ZIKV RNA on reverse-transcriptase polymerase-chain-reaction assay at 180 days, as well as terminal samples for other participants who submitted samples slightly more than 180 days after illness onset. Log 10 ZIKV RNA Load (copies/ml of semen) Culture Results Positive Negative Not cultured Days since Illness Onset Figure 1. Estimated Viral RNA Load and Culture Results in Semen Samples from 184 Men, Shown is the estimated viral RNA load and culture results for semen samples with Zika virus (ZIKV) RNA detected by reverse-transcriptase polymerasechain-reaction (RT-PCR) assay, according to days since illness onset, among 184 enrolled U.S. residents with symptomatic ZIKV infection in the period. Owing to specimen leakage during shipping or to inadequate volume, 97 samples could not be tested. Among the tested semen samples, 174 of 1230 (14%) were positive for ZIKV RNA according to RT-PCR. The highest viral RNA load detected in semen was 8.4 log 10 RNA copies per milliliter (Fig. 1). Overall, 60 of 184 men (33%) had at least 1 RNA-positive semen sample (range, 1 to 16; median, 3 positive samples per participant). The observed rates of viral RNA detection were similar in the subgroup of men who reported having undergone vasectomy, with ZIKV RNA detected in 34 of 201 samples (17%) that had been obtained from 7 of 28 men (25%) who had undergone vasectomy. The median viral RNA loads for the first positive sample were significantly lower in men who had undergone vasectomy than in men who had not, with a median of 3.6 log 10 RNA copies per milliliter versus 5.6 log 10 RNA copies per milliliter (χ 2 = 7.6, P = 06). Among men who submitted samples within 30 days after illness onset, 61% had detectable ZIKV RNA in their semen. This proportion decreased to 7% or less among men who submitted samples more than 90 days after illness onset (Table 2). The number of ZIKV RNA copies generally decreased in subsequent samples from the same man. However, the rate of decrease 1380 n engl j med 378;15 nejm.org April 12, 2018

5 Zika Virus Shedding in Semen varied greatly. Intermittent shedding, defined as a ZIKV RNA negative sample followed by a positive sample, was observed in 8 of 60 men (13%) (Fig. S2 in the Supplementary Appendix). On a per-sample basis, the frequency of a ZIKV RNA positive sample following a negative one was 1% (8 of 868 samples). Four men were enrolled for extended participation beyond the planned 6-month period. The longest duration of ZIKV RNA detection in semen was 281 days after illness onset in a man who had undergone vasectomy (Participant P28) (Fig. S2 in the Supplementary Appendix). Three of the four men reported that they did not have a known immunocompromising condition; information on immune status was not available for the fourth man. ZIKV RNA in Urine A total of 8 of 1038 urine samples (1%) submitted by 183 men yielded evidence of ZIKV RNA according to RT-PCR. The numbers of ZIKV RNA copies per milliliter of urine were lower than in semen, with a maximum level detected of 4.6 log 10 RNA copies per milliliter of urine and a median level of 2.9 log 10 RNA copies per milliliter. The 8 urine samples with ZIKV RNA detected were obtained from 7 men who had ZIKV RNA detected in contemporaneous semen samples; all 8 samples were obtained within 40 days after illness onset. Risk Factors for ZIKV RNA in Semen and Predicted Maximum Duration of Persistence Under the assumption of a Weibull distribution with averaging over all the covariate effects in the model, the mean time to the clearance of ZIKV RNA from semen was 54 days (95% confidence interval, 53 to 55). On the basis of our results, we estimated that only 5% of men would have detectable levels of ZIKV RNA at 158 days after the onset of symptoms (upper boundary of the 95% confidence interval, 186 days) and only 1% of men would have detectable levels of ZIKV RNA at 240 days after illness onset (upper boundary of the 95% confidence interval, 299 days) (Fig. 2). Factors that were independently associated with a longer time to RNA clearance from semen were increasing age (P = 1), symptoms at the time of initial illness (presence of conjunctivitis, P = 2; absence of joint pain, P = 03), and less frequent ejaculation (P = 02) (Fig. 3). On average, ZIKV RNA was detected 12 days longer in men who were 50 years of age than in those who were 30 years of age and 12 days longer in men who reported having had conjunctivitis during their initial illness than in those who did not. Men who reported no joint pain during their initial illness had ZIKV RNA detected 27 days longer than did men who reported joint pain. The largest overall effect, however, was that of ejaculatory frequency (Fig. 3B). After controlling for age, we found that men who ejaculated four times per week during the study cleared ZIKV RNA 21 days earlier than did men who ejaculated once per week. Among the 60 men who had at least one positive semen sample, the median ejaculatory frequency during shedding was nearly identical to that after shedding (see the Supplementary Appendix) Figure 2. Estimated Proportion of Semen Samples Positive for ZIKV on RT-PCR, According to Days since Illness Onset. Shown is the estimated proportion of semen samples that were positive for ZIKV on RT-PCR, according to the number of days since illness onset, evaluated as the average of all the covariate effects in the model. The solid black curve is the decline in the proportion of positive samples as estimated by the model. The dotted curve is the upper boundary of the 95% confidence interval of the decline. The horizontal dashed line at 1 indicates the estimated point at which the proportion of positive samples reached 1%. We estimate that only 1% of the participants would have had positive samples at 240 days. The upper boundary of the 95% confidence interval of this estimate is 299 days. n engl j med 378;15 nejm.org April 12,

6 The new england journal of medicine A ZIKV Clearance According to Age 20 yr of age 30 yr of age 40 yr of age 50 yr of age 60 yr of age B ZIKV Clearance According to Number of Ejaculations per Week 0 per wk 2 per wk 4 per wk 6 per wk 8 per wk 10 per wk 12 per wk C ZIKV Clearance According to Presence or Absence of Conjunctivitis D ZIKV Clearance According to Presence or Absence of Joint Pain Conjunctivitis No joint pain No conjunctivitis Joint pain Figure 3. Estimated Proportion of Semen Samples Positive for ZIKV on RT-PCR, According to Days since Illness Onset, for Independently Associated Covariates. Shown is the estimated proportion of semen samples that were positive for ZIKV on RT-PCR, at different time points after illness onset, according to each of four independently associated covariates: age of the participant (P = 1) (Panel A), mean frequency of ejaculation per week (P = 02) (Panel B), conjunctivitis during initial illness (P = 2) (Panel C), and absence of joint pain during initial illness (P = 03) (Panel D). The estimates were based on the testing of 1230 semen samples that had been obtained from 184 U.S. residents with symptomatic ZIKV infection. Isolation of ZIKV from Semen Among the semen samples in which ZIKV RNA was detected by RT-PCR, 78 samples that had been obtained from 46 men were tested by culture. Infectious ZIKV was detected by a plaque assay in 3 of 19 samples that were obtained within 30 days after illness onset but in none of 59 samples that were obtained later. The 3 samples that yielded infectious virus were obtained from 3 participants and were the only samples with at least 7.0 log 10 ZIKV RNA copies per milliliter of semen (Fig. 1). The presence of infectious ZIKV in the sample with 8.4 log 10 ZIKV RNA copies per milliliter was also shown by inoculation of the initial semen sample on Vero cells, followed by quantification of viral RNA in supernatant by RT-PCR at multiple times after inoculation. The ZIKV RNA copy number in these 3 samples was observed to increase over time. Infectious ZIKV was not detected in the next available sample from two of these participants; these samples were obtained 38 days and 59 days after illness onset and had ZIKV RNA copy numbers of 5.8 and 3.1 log 10 RNA copies per milliliter, respectively. The next available sample from the third participant was obtained 76 days after illness onset and had no detectable ZIKV RNA n engl j med 378;15 nejm.org April 12, 2018

7 Zika Virus Shedding in Semen ZIKV RNA was initially detected by RT-PCR in the cell-culture supernatant of 30 additional samples that had been inoculated onto Vero cells. However, copy numbers were lower than in the original sample and decreased over time, findings that suggest carryover of viral RNA from the original sample rather than the presence of infectious virus. Efforts to culture infectious ZIKV from the 8 urine samples with detectable ZIKV RNA were unsuccessful. Discussion In this study, more than 60% of men with symptomatic ZIKV infection were observed to have ZIKV RNA in their semen during the first 30 days after illness onset. The maximum viral load was 8.4 log 10 RNA copies per milliliter. ZIKV RNA shedding in semen decreased rapidly during the 3 months after the onset of illness. However, persistence for up to 9 months occurred in a small percentage of men. These observations are consistent with those in previous case reports and with interim results from smaller studies. 6,8,28,29 The results of multivariable analysis suggest that men who were older or who ejaculated infrequently were more likely to have prolonged shedding in semen than those who were younger or had more frequent ejaculation. The detection of ZIKV RNA in urine was rare in this study, probably because of the timing of the initial specimen collection. Previous studies have shown that shedding of ZIKV in urine, although initially common, declines rapidly during the first few weeks after illness onset. 29,30 In contrast to the frequent and prolonged shedding of ZIKV RNA in semen, the shedding of infectious ZIKV that could be cultured was rare, short-lived, and limited to the few samples with at least 7.0 log 10 ZIKV RNA copies per milliliter of semen. None of the 75 cultured samples with RNA loads lower than 7.0 log 10 per milliliter yielded infectious ZIKV, and no sample that was obtained more than 30 days after illness onset yielded infectious ZIKV. Similar findings have been reported in murine models. 31 Attempts by others to culture ZIKV from human semen at later time points have generally been unsuccessful. 9,32,33 In one case report, ZIKV RNA was detected in Vero cell cultures inoculated with semen that had been obtained 69 days after illness onset; it was unstated, however, whether the RNA copy number increased during culture. 34 In our study, culture supernatants often had detectable but declining ZIKV RNA copy numbers, which suggests passive carryover of RNA from the original inoculation rather than viral replication. The divergent results generated by culture and RT-PCR can be adjudicated by comparing them with epidemiologic data about known instances of sexual transmission. Among documented cases of male-to-partner sexual transmission, all have occurred within 41 days after illness onset in the source male partner, and most have occurred within 20 days. 7-9,12,13,35 The absence of reported events occurring at later time points suggests that transmission events coincide with the period during which the virus can be cultured and that detection of ZIKV RNA by RT-PCR may overstate the duration and magnitude of the risk of sexual transmission. Several factors appear to independently influence the rate at which ZIKV RNA is cleared from the semen of infected men. Prolonged shedding in older men may be related to either structural changes in the reproductive tract (e.g., prostatic hypertrophy) or perhaps reduced immunity. The observed associations with conjunctivitis and arthralgia may be markers of infectious or immunologic events also occurring in the testis or genitourinary mucosa, which in turn might affect the duration of shedding. Regarding the association between the duration of RNA shedding and ejaculatory frequency, one possible explanation is that men feel ill while shedding ZIKV and are therefore less likely to engage in sexual activity. If so, one would expect men s ejaculatory frequency to increase after shedding stopped. However, among men who had at least one positive semen sample, the mean ejaculatory frequency was the same during and after shedding. An alternative explanation is that participant-specific differences in ejaculatory frequency influence the rate at which viral material is flushed from the genital tract. The intermittent detection of ZIKV RNA in semen was noted in a few participants. This finding is consistent with previous reports 24,29 and suggests that a single negative result cannot rule out future shedding of ZIKV RNA. Nevertheless, the likelihood of detecting ZIKV RNA in semen when it was not detected in a previous sample appears to be less than 1%. Our findings are subject to several limitations. n engl j med 378;15 nejm.org April 12,

8 The new england journal of medicine They do not necessarily pertain to men with asymptomatic infection, although a recent study showed similar rates of ZIKV RNA shedding in semen of asymptomatic blood donors with viremia. 20 The clinical significance of low levels of ZIKV RNA in semen remains uncertain, and without access to medical records, it was not possible to evaluate the potential effect of underlying medical conditions on the duration of shedding. Some specimens could not be tested because of leakage, and the collection of samples at home and the delay associated with specimen transport and storage may have compromised the quality of other samples. Important questions regarding sexual transmission of ZIKV remain. Factors underlying the ZIKV tropism for human reproductive tissues have yet to be identified, 28,36 and although fetal infection has been reported after sexual or intravaginal ZIKV exposure in animal models 31,37 and possibly in humans, 38 it is unknown whether maternal infection through sex poses the same risks to the fetus as infection through mosquito bite. A better understanding of these issues is needed to guide the development of effective prevention strategies. The views expressed in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC). Supported by the CDC. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. We thank the study participants and our colleagues in state and local health departments and at the CDC, especially Alexander Davidson, Sally Slavinski, Marcy Layton, Charsey Cole Porse, Jacklyn Wong, Jennifer White, Mary Jo Polfleit, Katie Kurkjian, Hanna Oltean, Pat Ryan, Katherine Feldman, Connie Austin, Rebecca Osborn, Julia Jensen, Jennifer House, Taryn Stevens, Julie Shaffner, Rendi Murphree, Carina Blackmore, Sarah Y. Park, Amanda Feldpausch, Sara Robinson, Katie Brown, Randy Nelson, Abigail Mathewson, Carolyn Fredette, Miguella Mark- Carew, Carl Williams, Rachel Radcliffe, Alain Pujolar, Pauline Lucas, Natalie Kwit, Brad Biggerstaff, and Stacey Bartlett. References 1. Rasmussen SA, Jamieson DJ, Honein MA, Petersen LR. Zika virus and birth defects reviewing the evidence for causality. N Engl J Med 2016; 374: Dos Santos T, Rodriguez A, Almiron M, et al. Zika virus and the Guillain Barré syndrome case series from seven countries. N Engl J Med 2016; 375: Petersen LR, Jamieson DJ, Powers AM, Honein MA. Zika virus. N Engl J Med 2016; 374: Baud D, Gubler DJ, Schaub B, Lanteri MC, Musso D. An update on Zika virus infection. Lancet 2017; 390: Foy BD, Kobylinski KC, Chilson Foy JL, et al. Probable non-vector-borne transmission of Zika virus, Colorado, USA. Emerg Infect Dis 2011; 17: Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg Infect Dis 2015; 21: Hills SL, Russell K, Hennessey M, et al. Transmission of Zika virus through sexual contact with travelers to areas of ongoing transmission continental United States, MMWR Morb Mortal Wkly Rep 2016; 65: D Ortenzio E, Matheron S, Yazdanpanah Y, et al. Evidence of sexual transmission of Zika virus. N Engl J Med 2016; 374: Frank C, Cadar D, Schlaphof A, et al. Sexual transmission of Zika virus in Germany, April Euro Surveill 2016; 21: Fréour T, Mirallié S, Hubert B, et al. Sexual transmission of Zika virus in an entirely asymptomatic couple returning from a Zika epidemic area, France, April Euro Surveill 2016; 21: Turmel JM, Abgueguen P, Hubert B, et al. Late sexual transmission of Zika virus related to persistence in the semen. Lancet 2016; 387: Venturi G, Zammarchi L, Fortuna C, et al. An autochthonous case of Zika due to possible sexual transmission, Florence, Italy, Euro Surveill 2016; 21: Prevention of sexual transmission of Zika virus. Interim guidance update. Geneva: World Health Organization, September 2016 ( iris/ handle/ 10665/ ). 14. World Health Organization. Situation report. March 10, 2017 ( emergencies/ zika-virus/ situation-report/ en/). 15. Deckard DT, Chung WM, Brooks JT, et al. Male-to-male sexual transmission of Zika virus Texas, January MMWR Morb Mortal Wkly Rep 2016; 65: Davidson A, Slavinski S, Komoto K, Rakeman J, Weiss D. Suspected femaleto-male sexual transmission of Zika virus New York City, MMWR Morb Mortal Wkly Rep 2016; 65: Brooks RB, Carlos MP, Myers RA, et al. Likely sexual transmission of Zika virus from a man with no symptoms of infection Maryland, MMWR Morb Mortal Wkly Rep 2016; 65: Rowland A, Washington CI, Sheffield JS, Pardo-Villamizar CA, Segars JH. Zika virus infection in semen: a call to action and research. J Assist Reprod Genet 2016; 33: Moreira J, Peixoto TM, Siqueira AM, Lamas CC. Sexually acquired Zika virus: a systematic review. Clin Microbiol Infect 2017; 23: Musso D, Richard V, Teissier A, et al. Detection of Zika virus RNA in semen of asymptomatic blood donors. Clin Microbiol Infect 2017; 23(12): 1001.e e Brooks JT, Friedman A, Kachur RE, LaFlam M, Peters PJ, Jamieson DJ. Update: interim guidance for prevention of sexual transmission of Zika virus United States, July MMWR Morb Mortal Wkly Rep 2016; 65: Oster AM, Russell K, Stryker JE, et al. Update: interim guidance for prevention of sexual transmission of Zika virus United States, MMWR Morb Mortal Wkly Rep 2016; 65: Nicastri E, Castilletti C, Liuzzi G, Iannetta M, Capobianchi MR, Ippolito G. Persistent detection of Zika virus RNA in semen for six months after symptom onset in a traveller returning from Haiti to Italy, February Euro Surveill 2016; 21: Barzon L, Pacenti M, Franchin E, et al. Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January Euro Surveill 2016; 21: Zika virus disease, non-congenital infection and Zika virus, nongenital infection 2016 interim case definition, approved February 26, Atlanta: Council of State and Territorial Epidemiologists, 2016 ( / wwwn.cdc.gov/ nndss/ conditions/ zika/ case-definition/ 2016/ 02/ 26/ ) n engl j med 378;15 nejm.org April 12, 2018

9 Zika Virus Shedding in Semen 26. Weger-Lucarelli J, Duggal NK, Bullard- Feibelman K, et al. Erratum for Weger- Lucarelli et al., Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone. J Virol 2017; 91(8): e Jang HC, Park WB, Kim UJ, et al. First imported case of Zika virus infection into Korea. J Korean Med Sci 2016; 31: Mansuy JM, Dutertre M, Mengelle C, et al. Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen? Lancet Infect Dis 2016; 16: Paz-Bailey G, Rosenberg ES, Doyle K, et al. Persistence of Zika virus in body fluids preliminary report. N Engl J Med. DOI: /NEJMoa Bingham AM, Cone M, Mock V, et al. Comparison of test results for Zika virus RNA in urine, serum, and saliva specimens from persons with travel-associated Zika virus disease Florida, MMWR Morb Mortal Wkly Rep 2016; 65: Duggal NK, Ritter JM, Pestorius SE, et al. Frequent Zika virus sexual transmission and prolonged viral RNA shedding in an immunodeficient mouse model. Cell Rep 2017; 18: Froeschl G, Huber K, von Sonnenburg F, et al. Long-term kinetics of Zika virus RNA and antibodies in body fluids of a vasectomized traveller returning from Martinique: a case report. BMC Infect Dis 2017; 17: Matheron S, d Ortenzio E, Leparc- Goffart I, Hubert B, de Lamballerie X, Yazdanpanah Y. Long-lasting persistence of Zika virus in semen. Clin Infect Dis 2016; 63: Arsuaga M, Bujalance SG, Díaz-Menéndez M, Vázquez A, Arribas JR. Probable sexual transmission of Zika virus from a vasectomised man. Lancet Infect Dis 2016; 16: Harrower J, Kiedrzynski T, Baker S, et al. Sexual transmission of Zika virus and persistence in semen, New Zealand, Emerg Infect Dis 2016; 22: Govero J, Esakky P, Scheaffer SM, et al. Zika virus infection damages the testes in mice. Nature 2016; 540: Yockey LJ, Varela L, Rakib T, et al. Vaginal exposure to Zika virus during pregnancy leads to fetal brain infection. Cell 2016; 166(5): e Oliveira DB, Almeida FJ, Durigon EL, et al. Prolonged shedding of Zika virus associated with congenital infection. N Engl J Med 2016; 375: Copyright 2018 Massachusetts Medical Society. n engl j med 378;15 nejm.org April 12,