Prolonged Detection of Zika Virus in Vaginal Secretions and Whole Blood

Kristy O. Murray; Rodion Gorchakov; Anna R. Carlson; Rebecca Berry; Lilin Lai; Muktha Natrajan; Melissa N. Garcia; Armando Correa; Shital M. Patel; Kjersti Aagaard; Mark J. Mulligan


Emerging Infectious Diseases. 2016;23(1) 

In This Article

The Study

This study was reviewed and approved by the Baylor College of Medicine Institutional Review Board (H-30533). A previously healthy, nonpregnant, 26 year-old non-Hispanic white woman returned to the United States from Tegucigalpa, Honduras, during mid-May 2016. Five days after her return (day 0), signs and symptoms consistent with Zika virus infection developed, beginning with rash and subsequent fever, headache, and conjunctivitis ( Table ). Fever and rash continued through day 5 and day 6, respectively. By day 15, desquamation was noted on the palms of both hands and soles of both feet. By day 17, all symptoms had resolved.

Serial specimens were longitudinally collected for >11 weeks. The first specimens were collected on day 0, two hours after onset of rash and 2 h before development of fever. All remaining specimens were collected at 3, 8, 14, 21, 28, 35, 42, 53, 64, and 81 days after onset of illness. Specimens included serum, whole blood (EDTA anticoagulated), urine, saliva, and vaginal mucosa swabs. The patient was not menstruating when vaginal swab specimens were collected.

RNA was extracted from serum, whole blood, and urine samples by using the QIAamp MinElute Virus Spin Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. Oral and vaginal mucosal swab specimens were collected by using the BBL CultureSwab Collection and Transport System (Becton Dickinson, Franklin Lakes, NJ, USA). Specimens were incubated in 250 µL of AL/carrier RNA lysis buffer for 10 min at room temperature; 200 µL of phosphate-buffered saline was added before RNA extraction.

Eluted RNA from all samples was tested in a quantitative reverse transcription quantitative PCR (qRT-PCR) that included a TaqMan Fast Virus 1-Step Master Mix (ThermoFisher Scientific, Foster City, CA, USA) and a TaqMan ZIKV 1107 assay[6] with appropriate positive and negative controls. We detected Zika virus RNA in serum up to day 8 after onset of illness and in body fluids up to day 14; whole blood samples remained positive up to day 81 (Figure). Results of qRT-PCR of saliva were negative after day 8, and results for urine and vaginal swab specimens did not become negative until after day 14. We tested a day 0 serum sample for dengue virus and chikungunya virus RNA by using TaqMan assays;[7,8] all results were negative.


Quantitative reverse transcription PCR cycle threshold values over time (days after onset of illness) for whole blood, serum, urine, saliva, and vaginal mucosal swab specimens obtained from a 26-year-old woman infected with Zika virus who returned from Honduras to the United States. A cutoff value for a negative result was established at a cycle threshold of 40 (black horizontal line).

Virus isolations were performed for Vero cells in complete Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum. Cells were infected with day 0 serum samples (or mock-infected with cell culture medium) and observed for cytopathic effects. Cell culture supernatants were sampled 13 days after cell culture infection, and RNA was extracted and tested for Zika virus RNA. Supernatant was collected on day 14, and viral titer was 8.5 × 105 PFU/mL by plaque assay. Attempts to isolate virus from the day 64 erythrocyte fraction showed no evidence of cytopathic effects, and first and second passages were negative by qRT-PCR. Because the day 81 whole blood specimen was still positive by qRT-PCR, we used ficoll to separate peripheral blood mononuclear cells and erythrocytes and found that erythrocytes were the only fraction positive for Zika virus RNA. The partial sequence of the virus we isolated was submitted to GenBank under accession no. KX928077.

On day 8, plasma was evaluated by using an ELISA[9] to assess IgM and IgG binding to Zika virus envelope protein (Zika Virus Envelope Recombinant Protein, #R01635; Meridian Life Sciences, Memphis, TN, USA); positive results were obtained. Plasma-neutralizing antibodies against Zika virus were detected (50% focus reduction neutralization test titer 1:1,438), but neutralization of dengue virus serotypes 1–4 was not detected. These findings indicated a robust Zika virus–specific humoral response.