Neuroteratogenic Viruses: ZIKV, the placenta and IUGR

Following the emergence of Zika Virus (ZIKV) in the Americas in 2015, Brazil became the first country to report an increase in cases of microcephaly, followed by other countries including Colombia and the USA and ZIKV associated cases of foetal and neonatal microcephaly have been reported since elsewhere. ZIKV therefore joins five other neuroteratogenic viruses that cause neurological disorders in humans in addition to other neuroteratogenic viruses which have been associated with abnormal neurodevelopment in animals.

In humans, the most common infectious neuroteratogenic agents are summarised by the mnemonic “TORCH(S)” (Toxoplasmosis, Others, Rubella, (Human) Cytomegalovirus, Herpes Simplex, Syphilis) with ZIKV either classified as “Others” or by expanding the mnemonic to TORCHSZ and indeed health authorities in affected countries regularly test cases of microcephaly suspected to be associated with neurological infections not only for ZIKV but also for the presence of TORCH(S). Microcephaly however is only one outcome of CNS defects that are associated with the infection of foetal neural cells with infectious agents, as illustrated by the wide range of symptoms of neonate that include hydrocephalus, cerebellar dysplasia, hypomyelinogenesis or microphthalmia.

Common to all pathogens is, that they infect the foetus in utero rather than intra partum or vertical by breastfeeding, suggesting that the infection of neural precursor or mature neurons during the development of the foetal brain causes abnormal foetal brain development.  Therefore, it is crucial to examine both the ability of neuroteratogenic viruses to infect and replicate in vaginal tissue as well to examine the mechanism of transplacental transmission and thus infection of embryonic and/or foetal cells.

Table: Neuroteratogenic viruses 

In the case of ZIKV, the intra vaginal infection of wt, Irf-3 Irf-7 -/-, and Ifnar-1 -/+ pregnant mice with ZIKV FSS13025 is followed by transplacental transmission of ZIKV with the offspring of infected mice displaying signs of intrauterine growth restriction (IUGR). IUGR, signs of microcephaly including extensive apoptosis of cortical progenitor cells have also been demonstrated in the offspring of mice infected i.p. with ZIKV BR, suggesting that ZIKV indeed can cross the placenta. Studies in foetal mice displaying IUGR however frequently display neuronal cells that undergo virus independent apoptosis, i.e. the absence of viral antigen in neuronal cells undergoing apoptosis, suggesting that ZIKV infection can also cause bystander apoptosis and/or tissue injury independent of the initial viral cytopathic effect. Indeed, a recent study suggests that the infection of cranial crest neuronal cells (CNCC) with ZIKV MR766 only induces limited apoptosis of infected CNCC with extensive apoptosis of non-infected cells by inducing the expression of inflammatory cytokines thus inducing T lymphocyte dependent cytolysis as well as apoptosis of neighbouring neuronal cells, which might explain the intracranial calcifications that are commonly seen in cases of ZIKV associated microcephaly.

Transplacental transmission of ZIKV

The placenta constitutes the principal barrier between the viraemic mother and the non-infected embryo/foetus. In both humans and mice, chorionic villi are separated from the maternal blood by layers of placental multinucleate syncytia of trophoblast cells (PTB or STB) and Cytotrophoblasts(CTB), either by one cell layer (hemomonochorial; primate) or multiple cell layers (hemotrichorial; mouse), whereas in pigs the chorionic villi are separated from the maternal blood by the uterine epithelium, endothelium and connective tissue.

In primates, mononuclear extravillious trophoblasts (distally) extent into the decidua and are thus being exposed to maternal fibroblasts and leukocytes. The transmigration of leukocytes however is prevented due to the absence of intercellular junctions of the epithelium. In addition, STB are also expressing high levels of TLR and thus are capable of inducing a strong antiviral response as evidenced by high levels of Interferon-delta type 1 (IFN-𝚫1) infected with ZIKV FSS 13025 or ZIKV MR766 as well as potentially inducing the degradation of endosomes that contain ZIKV particles by autophagy following viral entry.  As mentioned above and as discussed in a previous post, ZIKV can infect and replicate in cells of the vaginal mucosa. It is therefore possible that the secretion of inflammatory cytokines can disrupt the placental barrier and thus infect foetal cells. Alternatively, ZIKV –and other neuroteratogenic viruses- may infect foetal M2 macrophages (Hofbauer cells) that express Fcg receptors as well as syncytiotrophoblasts (STB) that express foetal/neonatal Fc receptors (FcRn). This notion is supported by findings that ZIKV PR 2015 infects both Hofbauer cells and STB, concomitant with the expression of IFN-a and CXLB.

To investigate if placental tissue supports the replication of ZIKV and thus can transmit ZIKV to foetal tissue, organotypic cultures of chorionic or chorionic villus explants derived from human placental tissues obtained at different times gestation or Amniotic Epithelial Cells (AmEpC) and other cell lines including Human Placental Fibroblasts (HPF), Trophoblast Progenitor Cells (TBPC), and Chorionic Trophoblasts (CTB) obtained from foetal membranes were infected with different ZIKV strains, namely ZIKV Nica 1-16 (Nicaragua), ZIKV Nica 2-16 (Nicaragua), ZIKV MR766 (Uganda), ZIKV SBH2015 (Brazil), ZIKV PRVABC59 (Puerto Rico) or ZIKV FSS13025 (Cambodia) with viral replication being detected by immunofluorescence analysis for the viral E glycoprotein and the viral nonstructural NS3 protein as well as determining viral titres by plaque and focus forming assays.

AmEpC derived from mid- and late- gestation as well as CTB, TBPC, HPF or Human Umbilical Cord Vein Endothelial Cells (HUVEC) infected with either ZIKV MR766 or ZIKV Nica 1-16 stained positive both for NS3 and E protein at 72 hrs p.i. indicating that both ZIKV MR766 and ZIV Nica 1-16 replicate in cells derived from the human placenta. Furthermore, viral replication was accompanied by the release of viral particles, although the highest titres were obtained from AmEpC derived from the human placenta mid gestation infected with ZIKV MR766, ZIKV Nica 1-16 or ZIKV Nica 2-16 strains when compared to HPF, CTB, TBPC or HUVEC, which confirms previous reports that ZIKV PR 2015 can infect CTB derived from full term (> 37 weeks) human placentas.

Similar to CTB or TBCP, ZIKV MR766 and ZIKV FSS13025 can infect and replicate in Placental Trophoblast Cell lines such as JEG-3 and HTR-8 although these ZIKV strains do not replicate in Primary Human Trophoblast Cells (PHT) isolated from full term placental tissue, indicating that the ability to infect placental tissue with ZIKV varies during different stages of the pregnancy as well as between cell types. Indeed, the expression levels of the main ZIKV receptor Axl and a co-receptor, TIM-1, in AmEpC isolated from mid- to late gestation is strong with Axl being located at the plasma membrane during mid-gestation and vesicular during the late stages of gestation, indicating that viral entry might be inhibited during the later stages of gestation. In TBPC, TIM-1 -a potential ZIKV co-receptor- is expressed both at 7.3 and 15.6 weeks gestational stage, whereas the expression of Axl is increased at week 15.6. Primary CTB however do not express Axl at late stages of gestation (in contrast to mid stage gestation), suggesting that at this point ZIKV entry might be primarily mediated by TIM-1.

Interestingly, ZIKV MR766 infected CTB cease to proliferate as measured by Ki-67 staining, suggesting that the infection of CTB with ZIKV MR766 induces a cell cycle arrest, which might be accompanied by the induction of apoptosis or senescence. In contrast to CTB, syncytiotrophoblasts (STB) do not support viral replication although the viral E protein can be detected in cytoplasmic vesicles in ZIKV Nica 1-16 infected indicating that viral replication might be inhibited either post-entry by preventing the release of the viral genome or by degradation of de novo synthesized viral proteins probably due to high levels of IFN-l1 as discussed above and in a previous post as well as the other factors such as IFITM-3.

In addition to AmEpC, CTB and STB, ZIKV has also been shown to infect primary decidual fibroblasts (dFibroblasts) as demonstrated by high viral titres of  and the presence of ZIKV E protein in dFibroblasts infected with a ZIKV strain derived from a French patient that got infected in Brazil (ZIKV FR) 72 hrs p.i..In addition to dFibroblasts, decidual Macrophages are also susceptible to ZIKV FR, supporting observations that ZIKV PR 2015 can infect and replicate in placental macrophages (Hofbauer cells).

In conclusion, ZIKV MR766 and ZIKV Nica1/2-2016 isolates have been demonstrated to productively infect Amniotic Cell Epithelial Cells (AmEpC), Trophoblast Progenitor Cells (TBPC), Chorionic Trophoblasts (CTB), human placental/decidual Fibroblast cells (HPF/dFibroblasts), decidual Macrophages and Hofbauer cells particularly if these cells were obtained from placental tissue early or mid-stage gestation and thus allow the infection of foetal neuronal progenitor cells or foetal macrophages. Decidual macrophages play a key role in regulating the vascular remodelling during pregnancy and secrete a wide range of cytokines including Interleukin-1β, -2, -4, -5, -6, -8, -10, -13 and TNF-α as well as proteases (matrix metalloproteinase-1, -2, -7, -9, and -10). The infection of dMacrophages therefore might induce inflammation and tissue damage following ZIKV infection as well as abnormal vascular development of the human placenta, thus be at least a contributing factor in the development of IUGR and potentially in ZIKV associated miscarriages independent of abnormal neural development.

Moreover, the infection of Fcg receptor (Fcg R) bearing foetal STB or CTB as well as maternal cells of the basal and parietal decidua including Hofbauer cells might be enhanced by transcytosis of IgG-ZIKV complexes consisting of DENV cross reactive antibodies and/or maternal ZIKV antibodies and ZIKV particles.  In this instance, the IgG-ZIKV complex enters the cell via phagocytosis of the complex after the activation of the FcgR which induces the phosphorylation of the immunoreceptor tyrosine based activation motif (ITAM), thus allowing the recruitment of Zap70 and Syk family proteins and activation of downstream pathways (including MAPK, PI3-K,PLCg, Rho, and Rac). These downstream pathways in turn contribute to vascular injury due to a “cytokine storm”, the release of high levels of pro-inflammatory cytokines, thus potentially contributing to the development of IUGR rather than abnormal brain development. Further experimental data are however needed to confirm this hypothesis.

The uptake of ZIKV into macrophages might also be stimulated by autophagy similar to antibody enhanced uptake of DENV into human pre-basophil/Mast cells.In this case, sub-neutralising antibodies from a prior infection stimulate the uptake of IgG-DENV complexes in both KU812 and HMC-1 cells which can be inhibited by either 3-MA or the expression of a catalytically inactive mutant of Atg4B, Atg4B^C74A. It should be noted however that it has not be conclusively proven that autophagy is required for viral uptake; based on the published results, it cannot be ruled out that the IgG-DENV complex increases viral entry via an autophagy independent mechanism and that the inhibition of viral replication by 3-MA or Atg4B^C74A  occurs by inhibiting the formation of viral replication centres. and/or by inhibiting the antiviral interferon type I signalling pathway. 

Further reading

Kim K, & Shresta S (2016). Neuroteratogenic Viruses and Lessons for Zika Virus Models. Trends in microbiology, 24 (8), 622-36 PMID: 27387029 

Yockey LJ, Varela L, Rakib T, Khoury-Hanold W, Fink SL, Stutz B, Szigeti-Buck K, Van den Pol A, Lindenbach BD, Horvath TL, & Iwasaki A (2016). Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection. Cell, 166 (5), 1247-12560000 PMID: 27565347 

  

Bayless NL, Greenberg RS, Swigut T, Wysocka J, & Blish CA (2016). Zika Virus Infection Induces Cranial Neural Crest Cells to Produce Cytokines at Levels Detrimental for Neurogenesis. Cell host & microbe, 20 (4), 423-428 PMID: 27693308 

El Costa H, Gouilly J, Mansuy JM, Chen Q, Levy C, Cartron G, Veas F, Al-Daccak R, Izopet J, & Jabrane-Ferrat N (2016). ZIKA virus reveals broad tissue and cell tropism during the first trimester of pregnancy. Scientific reports, 6 PMID: 27759009 

Tabata T, Petitt M, Puerta-Guardo H, Michlmayr D, Wang C, Fang-Hoover J, Harris E, & Pereira L (2016). Zika Virus Targets Different Primary Human Placental Cells, Suggesting Two Routes for Vertical Transmission. Cell host & microbe, 20 (2), 155-66 PMID: 27443522 

Quicke KM, Bowen JR, Johnson EL, McDonald CE, Ma H, O'Neal JT, Rajakumar A, Wrammert J, Rimawi BH, Pulendran B, Schinazi RF, Chakraborty R, & Suthar MS (2016). Zika Virus Infects Human Placental Macrophages. Cell host & microbe, 20 (1), 83-90 PMID: 27247001 

Bayer A, Lennemann NJ, Ouyang Y, Bramley JC, Morosky S, Marques ET Jr, Cherry S, Sadovsky Y, & Coyne CB (2016). Type III Interferons Produced by Human Placental Trophoblasts Confer Protection against Zika Virus Infection. Cell host & microbe, 19 (5), 705-12 PMID: 27066743 

Ning F, Liu H, & Lash GE (2016). The Role of Decidual Macrophages During Normal and Pathological Pregnancy. American journal of reproductive immunology (New York, N.Y. : 1989), 75 (3), 298-309 PMID: 26750089 

Lash GE, Pitman H, Morgan HL, Innes BA, Agwu CN, & Bulmer JN (2016). Decidual macrophages: key regulators of vascular remodeling in human pregnancy. Journal of leukocyte biology, 100 (2), 315-25 PMID: 26819320 

Salamonsen LA, Hannan NJ, & Dimitriadis E (2007). Cytokines and chemokines during human embryo implantation: roles in implantation and early placentation. Seminars in reproductive medicine, 25 (6), 437-44 PMID: 17960528 

Olagnier D, Amatore D, Castiello L, Ferrari M, Palermo E, Diamond MS, Palamara AT, & Hiscott J (2016). Dengue Virus Immunopathogenesis: Lessons Applicable to the Emergence of Zika Virus. Journal of molecular biology, 428 (17), 3429-48 PMID: 27130436

Huang X, Yue Y, Li D, Zhao Y, Qiu L, Chen J, Pan Y, Xi J, Wang X, Sun Q, & Li Q (2016). Antibody-dependent enhancement of dengue virus infection inhibits RLR-mediated Type-I IFN-independent signalling through upregulation of cellular autophagy. Scientific reports, 6 PMID: 26923481 

Fang, Y., Wan, S., Lu, Y., Yao, J., Lin, C., Hsu, L., Brown, M., Marshall, J., Anderson, R., & Lin, Y. (2014). Autophagy Facilitates Antibody-Enhanced Dengue Virus Infection in Human Pre-Basophil/Mast Cells PLoS ONE, 9 (10) DOI: 10.1371/journal.pone.0110655