Probes for INS

Context .- The placenta is an important component in understanding the fetal response to intrauterine Zika virus infection, but the pathologic changes in this organ remain largely unknown. Hofbauer cells are fetal-derived macrophages normally present in the chorionic villous stroma. They have been implicated in a variety of physiological and pathologic processes, in particular involving infectious agents. Objectives .- To characterize the fetal and maternal responses and viral localization in the placenta following Zika virus transmission to an 11 weeks' gestation fetus. The clinical course was notable for prolonged viremia in the mother and extensive neuronal necrosis in the fetus. The fetus was delivered at 21 weeks' gestation after pregnancy termination. Design .- The placenta was evaluated by using immunohistochemistry for inflammatory cells (macrophages/monocytes [Hofbauer cells], B and T lymphocytes) and proliferating cells, and an RNA probe to Zika virus. The fetal brain and the placenta were previously found to be positive by reverse transcription-polymerase chain reaction for Zika virus RNA. Results .- The placenta demonstrated prominently enlarged, hydropic chorionic villi with hyperplasia and focal proliferation of Hofbauer cells. The degree of Hofbauer cell hyperplasia gave an exaggerated immature appearance to the villi. No acute or chronic villitis, villous necrosis, remote necroinflammatory abnormalities, chorioamnionitis, funisitis, or hemorrhages were present. An RNA probe to Zika virus was positive in villous stromal cells, presumably Hofbauer cells. Conclusions .- Zika virus placental infection induces proliferation and prominent hyperplasia of Hofbauer cells in the chorionic villi but does not elicit villous necrosis or a maternal or fetal lymphoplasmacellular or acute inflammatory cell reaction.

A major concern associated with ZIKV infection is the increased incidence of microcephaly with frequent calcifications in infants born from infected mothers. To date, postmortem analysis of the central nervous system (CNS) in congenital infection is limited to individual reports or small series. We report a comprehensive neuropathological study in ten newborn babies infected with ZIKV during pregnancy, including the spinal cords and dorsal root ganglia (DRG), and also muscle, pituitaries, eye, systemic organs, and placentas. Using in situ hybridization (ISH) and electron microscopy, we investigated the role of direct viral infection in the pathogenesis of the lesions. Nine women had Zika symptoms between the 4th and 18th and one in the 28th gestational week. Two babies were born at 32, one at 34 and 36 weeks each and six at term. The cephalic perimeter was reduced in four, and normal or enlarged in six patients, although the brain weights were lower than expected. All had arthrogryposis, except the patient infected at 28 weeks gestation. We defined three patterns of CNS lesions, with different patterns of destructive, calcification, hypoplasia, and migration disturbances. Ventriculomegaly was severe in the first pattern due to midbrain damage with aqueduct stenosis/distortion. The second pattern had small brains and mild/moderate (ex-vacuo) ventriculomegaly. The third pattern, a well-formed brain with mild calcification, coincided with late infection. The absence of descending fibres resulted in hypoplastic basis pontis, pyramids, and cortico-spinal tracts. Spinal motor cell loss explained the intrauterine akinesia, arthrogryposis, and neurogenic muscle atrophy. DRG, dorsal nerve roots, and columns were normal. Lympho-histiocytic inflammation was mild. ISH showed meningeal, germinal matrix, and neocortical infection, consistent with neural progenitors death leading to proliferation and migration disorders. A secondary ischemic process may explain the destructive lesions. In conclusion, we characterized the destructive and malformative consequences of ZIKV in the nervous system, as reflected in the topography and severity of lesions, anatomic localization of the virus, and timing of infection during gestation. Our findings indicate a developmental vulnerability of the immature CNS, and shed light on possible mechanisms of brain injury of this newly recognized public health threat.

Zika virus (ZIKV) infection during pregnancy leads to devastating fetal outcomes, including intrauterine growth restriction and microcephaly. Greater understanding of mechanisms underlying ZIKV maternal-fetal transmission is needed to develop new therapeutic interventions. Here, we define an important role for the autophagy pathway in ZIKV vertical transmission. ZIKV infection induced autophagic activity in human trophoblasts and pharmacological inhibition limited ZIKV infectivity. Furthermore, deficiency in an essential autophagy gene, Atg16l1, in mice limited ZIKV vertical transmission and placental and fetal damage and overall improved placental and fetal outcomes. This protection was due to a placental trophoblast cell-autonomous effect of autophagic activity, not to alterations in systemic maternal ZIKV infection. Finally, an autophagy inhibitor, hydroxychloroquine, approved for use in pregnant women, attenuated placental and fetal ZIKV infection and ameliorated adverse placental and fetal outcomes. Our study reveals new insights into the mechanism of ZIKV vertical transmission and suggests that an autophagy-based therapeutic warrants possible evaluation in humans to diminish the risks of ZIKV maternal-fetal transmission.

The common small animal disease models for Zika virus (ZIKV) are mice lacking the interferon responses, but infection of interferon receptor α/β knock out (IFNAR−/−) mice is not uniformly lethal particularly in older animals. Here we sought to advance this model in regard to lethality for future countermeasure efficacy testing against more recent ZIKV strains from the Asian lineage, preferably the American sublineage. We first infected IFNAR−/− mice subcutaneously with the contemporary ZIKV-Paraiba strain resulting in predominantly neurological disease with ~50% lethality. Infection with ZIKV-Paraiba by different routes established a uniformly lethal model only in young mice (4-week old) upon intraperitoneal infection. However, intraperitoneal inoculation of ZIKV-French Polynesia resulted in uniform lethality in older IFNAR−/− mice (10–12-weeks old). In conclusion, we have established uniformly lethal mouse disease models for efficacy testing of antivirals and vaccines against recent ZIKV strains representing the Asian lineage.

Zika virus (ZIKV) is an emerging flavivirus that caused thousands of human infections in recent years. Compared to other human flaviviruses, ZIKV replication is not well understood. Using fluorescent, transmission electron, and focused ion beam-scanning electron microscopy, we examined ZIKV replication dynamics in Vero 76 cells and in the brains of infected laboratory mice. We observed the progressive development of a perinuclear flaviviral replication factory both in vitro and in vivo. In vitro, we illustrated the ZIKV lifecycle from particle cell entry to egress. ZIKV particles assembled and aggregated in an induced convoluted membrane structure and ZIKV strain-specific membranous vesicles. While most mature virus particles egressed via membrane budding, some particles also likely trafficked through late endosomes and egressed through membrane abscission. Interestingly, we consistently observed a novel sheet-like virus particle array consisting of a single layer of ZIKV particles. Our study further defines ZIKV replication and identifies a novel hallmark of ZIKV infection.

Zika virus (ZIKV), which can cause devastating disease in fetuses of infected pregnant women, can be transmitted by mosquito inoculation and sexual routes. Little is known about immune protection against sexually transmitted ZIKV. In this study, we show that previous infection through intravaginal or subcutaneous routes with a contemporary Brazilian strain of ZIKV can protect against subsequent intravaginal challenge with a homologous strain. Both routes of inoculation induced high titers of ZIKV-specific and neutralizing antibody in serum and the vaginal lumen. Virus-specific T cells were recruited to and retained in the female reproductive tract after intravaginal and subcutaneous ZIKV infection. Studies in mice with genetic or acquired deficiencies in B and/or T cells demonstrated that both lymphocyte populations redundantly protect against intravaginal challenge in ZIKV-immune animals. Passive transfer of ZIKV immune IgG or T cells significantly limited intravaginal infection of naïve mice, although antibody more effectively prevented dissemination throughout the reproductive tract. Collectively, our experiments begin to establish the immune correlates of protection against intravaginal ZIKV infection, which should inform vaccination strategies in non-pregnant and pregnant women.IMPORTANCE The recent ZIKV epidemic resulted in devastating outcomes in fetuses and may affect reproductive health. Unlike other flaviviruses, ZIKV can be spread by sexual contact as well as a mosquito vector. While previous studies have identified correlates of protection for mosquito-mediated infection, few have focused on immunity against sexual transmission. As exposure to ZIKV via mosquito bite has likely occurred to many living in endemic areas, our study addresses whether this route of infection can protect against subsequent sexual exposure. We demonstrate that subcutaneous ZIKV infection can protect against subsequent vaginal infection by generating both local antiviral T cell and antibody responses. Our research begins to define the immune correlates of protection for ZIKV infection in the vagina and provides a foundation for testing ZIKV vaccines against sexual transmission