Zika Virus RNA Replication and Persistence in Brain and Placental Tissue

Julu Bhatnagar; Demi B. Rabeneck; Roosecelis B. Martines; Sarah Reagan-Steiner; Yokabed Ermias; Lindsey B.C. Estetter; Tadaki Suzuki; Jana M. Ritter; M. Kelly Keating; Gillian Hale; Joy Gary; Atis Muehlenbachs; Amy Lambert; Robert Lanciotti; Titilope Oduyebo; Dana Meaney-Delman; Fernando Bolaños; Edgar Alberto Parra Saad; Wun-Ju Shieh; Sherif R. Zaki

Disclosures

Emerging Infectious Diseases. 2017;23(3):405-414. 

In This Article

Abstract and Introduction

Abstract

Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients (brains from 8 infants with microcephaly and placental/fetal tissues from 24 women) were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections.

Introduction

Zika virus has recently caused global concern because of an unprecedented outbreak of infection in Brazil and its association with congenital microcephaly and other adverse pregnancy outcomes, including pregnancy loss.[1–4] Vertical transmission of Zika virus from infected mothers to fetuses has been reported.[5–7] However, the mechanism of intrauterine transmission of Zika virus, cellular targets of viral replication, and the pathogenesis that leads to microcephaly and other congenital malformations have not yet been completely elucidated.

Recent in vitro studies that used brain organoids, neurospheres, and human pluripotent stem cell–derived brain cells have demonstrated Zika virus infection of human neural stem and progenitor cells and have also shown that placental macrophages are permissive to Zika virus infection.[8–13] Several studies that used mouse models have revealed that Zika virus infection of mice during early pregnancy results in infection of placenta and fetal brain, causing intrauterine growth restrictions, spontaneous abortions, and fetal demise.[14–16] Animal models and in vitro studies, although providing valuable insights, might not exactly reflect Zika virus disease processes in humans.[9,17] We previously detected Zika virus antigens in placentas of women and in human fetal or neonatal brains.[18,19] However, the presence of antigens does not necessarily indicate virus replication. Previous case studies have detected Zika virus RNA by reverse transcription PCR (RT-PCR) in fetal or neonatal brains, in amniotic fluid, and in placentas of women who had acquired Zika virus infection during early pregnancy.[5,20–22] Nevertheless, localization of replicating Zika virus RNA directly in the tissues of patients with congenital and pregnancy-associated infections is critical for identifying cellular targets of Zika virus infection and virus persistence in various tissues and for further investigating the mechanism of Zika virus intrauterine transmission.

Furthermore, laboratory diagnosis of congenital and pregnancy-associated Zika virus infections, particularly those involving adverse pregnancy outcomes, is also challenging because of the typically short duration of viremia.[23,24] Generally, Zika virus RT-PCR can detect viral RNA in serum within 3–10 days of symptom onset.[24,25] Thus, diagnosis by serum RT-PCR can be difficult for neonates who acquire Zika virus infection in utero and for women who acquire (undiagnosed) Zika virus infection during early pregnancy and later experience adverse pregnancy or birth outcomes, because Zika virus RNA generally clears from maternal/infant serum by the time the infant is born or infection is suspected. Serologic testing by ELISA, along with plaque-reduction neutralization testing, can be useful for these cases but may not always provide conclusive Zika virus diagnosis for patients with previous flavivirus exposure or immunization[23–25] and cannot characterize the virus strain and genotype. As a part of the ongoing Zika virus public health response, we developed Zika virus RT-PCR and in situ hybridization (ISH) assays for the detection and localization of Zika virus RNA in formalin-fixed, paraffin-embedded (FFPE) tissues and tested various tissues from infants with microcephaly who died. We also tested placental/fetal tissues from a series of women suspected of being infected with Zika virus during various stages of pregnancy.

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