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newborns from mothers infected during pregnancy. Congenital Zika syndrome refers to a constellation of neurological pathologies associated with ZIKV infection which may include microcephaly, reduced cerebral volume, ventriculomegaly (dilated lateral ventricles), cerebellar hypoplasia (incomplete development of 1 the cerebellum), lissencephaly (smooth Nicholas J.C. King, Mauro 2 brain cortex without gyri), and arthrogryM. Teixeira, and posis (congenital joint contractures) [2]. 3, Suresh Mahalingam * Active viral infection has been demonstrated in the placenta and brain of the Immune status changes during preterm fetus from ZIKV-infected pregnancy, with pro-inflammatory mothers.
Zika Virus: Mechanisms of Infection During Pregnancy
and anti-inflammatory contexts at different stages, [30_TD$IF]making pregnant women potentially more susceptible to various infections. Infection by Zika virus during pregnancy can cause developmental damage to the fetus, and the altered immune response during pregnancy could contribute to disease during Zika infection. It is well recognized that immune status changes during pregnancy. Originally thought to be a period of immunosuppression to ensure survival of the semiallogeneic fetus, this view shifted to one of an anti-inflammatory, Th2 skewing of the Th1/Th2 axis. However, current evidence supports the development of both proinflammatory and anti-inflammatory environments at different stages of pregnancy, making the materno-fetal interface central in the modulation and control of immune responses during gestation. Many viral infections, for example, influenza, hepatitis E, herpes simplex, measles, and smallpox, cause more severe disease in pregnancy, raising the question of whether pregnant women are more susceptible to infection at particular stages of gestation [1]. In the last 2 years it has become clear that Zika virus (ZIKV) causes developmental damage (congenital Zika syndrome), evident in a significant proportion of
As with known teratogenic viruses, such as rubella, ZIKV infection of the embryo is more likely to result in severe developmental damage with infection in the first few months of pregnancy, that is, during the rapid, early development of the brain. However, fetal infection may occur at any stage of pregnancy, and in the absence of microcephaly[31_TD$IF], babies born to ZIKVinfected mothers may nevertheless manifest morphological changes identifiable by neuroimaging [3]. Furthermore, since the brain is not fully developed even at birth, more subtle neurological damage may only become evident later in delayed developmental milestones. As more children are born in areas where ZIKV epidemics have occurred, and diagnostic tests become better able to differentiate ZIKV from other flavivirus infections, especially dengue, a more complete clinical picture of the outcomes associated with congenital ZIKV infection will undoubtedly emerge. How the fetus is infected by ZIKV is fundamental to informing interventional approaches. Studies in experimental animals confirm that infection during the early stages of pregnancy is more likely to cause the most severe forms of the congenital disease [32_TD$IF][4]. However, it is clear that infection at later stages may also cause fetal disease [32_TD$IF][4,2]. In endemic areas, most pregnant women become infected from the bite of an infected
mosquito. Vaginal inoculation through sexual contact with an infected male is clearly an important form of transmission in nonendemic areas, but its relevance in endemic regions remains unclear [3_TD$IF][5]. While antibodies from previous dengue infections may cause enhancement of ZIKV infection in vitro, the role of previous infections in causing congenital disease remains to be determined. The latter has often been raised to explain the apparent greater frequency of severe congenital Zika syndrome in the Northeast of Brazil. On the other hand, one study in Brazil has shown that areas with lower yellow fever vaccination coverage were more likely to have cases of microcephaly [34_TD$IF][6]. Thus, more detailed studies are needed to determine the relevance of previous and/or concomitant arboviral infections or vaccinations to ZIKV-associated congenital syndromes. Several studies have now evaluated pathological changes in the placenta associated with ZIKV infection in the fetus. Overall, it is clear that maternal ZIKV infection during pregnancy causes placental infection, with significant levels of virus in placental macrophages (Hofbauer cells) and placental villous fibroblasts [35_TD$IF][7,8]. In addition, most studies have shown that fetuses with significant damage have viral RNA or protein in the brain. Therefore, although local placental changes may contribute to fetal disease pathogenesis, it seems that the virus must reach the fetal brain for ensuing central nervous system damage to occur. How does ZIKV reach the developing fetal brain? Virus in the maternal blood must be transmitted across the placenta, in particular, the syncytiotrophoblast. This first barrier is syncytial, capable of producing type I and III interferons. These cells are highly resistant to infection, at least at the time of parturition in humans [36_TD$IF][9], although it is unclear if the levels of type III interferon required to maintain this resistance (which would also likely protect local placental cells from infection) are sustained from
Trends in Microbiology, September 2017, Vol. 25, No. 9
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early in pregnancy. A recent study has shown that cells from human term placentas, which resist infection, expressed genes associated with antiviral defense but not genes that encode attachment factors linked to ZIKV entry. In contrast, trophoblasts derived from embryonic stem cells lacked strong innate antiviral responses and also expressed attachment factors for ZIKV entry [37_TD$IF][10]. AXL and TIM1, putative receptors for ZIKV, are expressed by cells throughout the placenta and amniotic epithelium, including Hofbauer cells, making these cells susceptible to ZIKV infection in vitro [38_TD$IF][8]. These motile placental macrophages may thus function as Trojan horses, spreading the virus to the rest of the placenta, which in turn may facilitate transmission into the fetal circulation. Viral replication in placental macrophages induced type I IFN, proinflammatory cytokines, and antiviral gene expression, with relatively little cell death [39_TD$IF][7]. However, high levels of pro-inflammatory cytokine expression secondary to infection can affect fetal development, as emphasized by recent findings that cell damage in the developing brain, including premature maturation and apoptosis, is mediated by cytokine outputs from nearby ZIKV-infected cells [40_TD$IF][11]. Experimentally, mice show an early ZIKV-susceptible teratogenic window between gastrulation and organogenesis, suggesting infection of the embryo prior to full placentation. Infection later in gestation resulted in morphologically normal fetuses in about half of the conceptuses and containment of ZIKV within the placenta. However, where fetuses were abnormal, significant placental vascular disruption was also evident, suggesting that impaired materno-fetal circulation also contributes to pathology [32_TD$IF][4]. Unlike most other mammals, mice have a hemochorial placenta, similar to humans. Notwithstanding several anatomical
702
differences – for example, mice have three trophoblast layers (hemotrichorial) separating maternal and fetal circulations, compared to just one ultimately (hemomonochorial) in humans – the direct contact of the trophoblast with the maternal blood circulation in both species makes the mouse a useful experimental approximation of human placental virus transmission. Once in the fetus, and subsequently in the developing fetal brain, ZIKV infection corresponds [41_TD$IF]to the expression of AXL on neural progenitors and glial cells [42_TD$IF][12], although Retallack et al. reported [43_TD$IF]the relative resistance of mature neurons to infection and the susceptibility of glial cells in the same organotypic cultures [4_TD$IF][13]. This is the reverse of the in vivo situation for neurotropic flaviviruses, which tend to infect only neurons in adult humans and animal models. However, the early developmental stage of microglia in the embryo, making [45_TD$IF]these cells more susceptible to productive infection (rather than resistant, like adult microglia), the access by ZIKV to the brain parenchyma provided by the immaturity of the blood–brain barrier, and the ineffectual virus control by an immature fetal adaptive immune response may together permit a more generalized infection of the brain parenchyma in the embryo than in the adult. How the altered maternal immune response during pregnancy contributes to disease during ZIKV infection is an important area for further investigation. Identifying immune markers associated with development of (or protection from) microcephaly and other central nervous system defects will be valuable in determining the key immune processes during infection. Some of these outcomes will be difficult or time-consuming to achieve in human patient populations [46_TD$IF]and we
Trends in Microbiology, September 2017, Vol. 25, No. 9
expect that insights from pregnant animal models of ZIKV infection will make major contributions to our understanding of disease in the next few years. 1 Discipline of Pathology, Bosch Institute, School of Medical Sciences, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School, Charles Perkins Centre, University of Sydney, Camperdown, NSW, Australia 2 Instituto de Ciencias Biologicas, Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais, Brazil 3 Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland 4222, Australia *Correspondence: s.mahalingam@griffith.edu.au (S. Mahalingam). http://dx.doi.org/10.1016/j.tim.2017.05.005 References 1. Kourtis, A.P. et al. (2014) Pregnancy and infection. N. Engl. J. Med. 370, 2211–2218 2. Melo, A.S. et al. (2016) Congenital Zika virus infection: beyond neonatal microcephaly. JAMA Neurol. 73, 1407–1416 3. van der Linden, V. et al. (2016) Description of 13 infants born during October 2015–January 2016 with congenital Zika virus infection without microcephaly at birth – Brazil. MMWR. Morb. Mortal. Wkly Rep. 65, 1343–1348 4. Xavier-Neto, J. (2017) Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: A developmental study. PLoS Negl. Trop. Dis. e0005363 5. Mansuy, J.M. et al. (2016) Zika virus: high infectious viral load in semen, a new sexually tansmitted pathogen? Lancet Infect. Dis. 16, 405 6. De Góes Cavalcanti, L.P. et al. (2016) Zika virus infection, associated microcephaly, and low yellow fever vaccination coverage in Brazil: is there any causal link? J. Infect. Dev. Ctries. 10, 563–566 7. Quicke, K.M. et al. (2016) Zika virus infects human placental macrophages. Cell Host Microbe 20, 83–90 8. Tabata, T. et al. (2016) Zika virus targets different primary human placental cells, suggesting two routes for vertical transmission. Cell Host Microbe 20, 155–166 9. Bayer, A. et al. (2016) Type III interferons produced by human placental trophoblasts confer protection against Zika virus infection. Cell Host Microbe 19, 705–712 10. Sheridan, M.A. et al. (2017) Vulnerability of primitive human placental trophoblast to Zika virus. Proc. Natl. Acad. Sci. U. S. A. 114, E1587–E1596 11. Bayless, N.L. et al. (2016) Zika virus infection induces cranial neural crest cells to produce cytokines at levels detrimental for neurogenesis. Cell Host Microbe 20, 423– 428 12. Nowakowski, T.J. et al. (2016) Expression analysis highlights AXL as a candidate Zika virus entry receptor in neural stem sells. Cell Stem Cell 18, 591–596 13. Retallack, H. et al. (2016) Zika virus cell tropism in the developing human brain and inhibition by azithromycin. Proc. Natl. Acad. Sci. U. S. A. 113, 14408–14413
newborns from mothers infected during pregnancy. Congenital Zika syndrome refers to a constellation of neurological pathologies associated with ZIKV infection which may include microcephaly, reduced cerebral volume, ventriculomegaly (dilated lateral ventricles), cerebellar hypoplasia (incomplete development of 1 the cerebellum), lissencephaly (smooth Nicholas J.C. King, Mauro 2 brain cortex without gyri), and arthrogryM. Teixeira, and posis (congenital joint contractures) [2]. 3, Suresh Mahalingam * Active viral infection has been demonstrated in the placenta and brain of the Immune status changes during preterm fetus from ZIKV-infected pregnancy, with pro-inflammatory mothers.
Zika Virus: Mechanisms of Infection During Pregnancy
and anti-inflammatory contexts at different stages, [30_TD$IF]making pregnant women potentially more susceptible to various infections. Infection by Zika virus during pregnancy can cause developmental damage to the fetus, and the altered immune response during pregnancy could contribute to disease during Zika infection. It is well recognized that immune status changes during pregnancy. Originally thought to be a period of immunosuppression to ensure survival of the semiallogeneic fetus, this view shifted to one of an anti-inflammatory, Th2 skewing of the Th1/Th2 axis. However, current evidence supports the development of both proinflammatory and anti-inflammatory environments at different stages of pregnancy, making the materno-fetal interface central in the modulation and control of immune responses during gestation. Many viral infections, for example, influenza, hepatitis E, herpes simplex, measles, and smallpox, cause more severe disease in pregnancy, raising the question of whether pregnant women are more susceptible to infection at particular stages of gestation [1]. In the last 2 years it has become clear that Zika virus (ZIKV) causes developmental damage (congenital Zika syndrome), evident in a significant proportion of
As with known teratogenic viruses, such as rubella, ZIKV infection of the embryo is more likely to result in severe developmental damage with infection in the first few months of pregnancy, that is, during the rapid, early development of the brain. However, fetal infection may occur at any stage of pregnancy, and in the absence of microcephaly[31_TD$IF], babies born to ZIKVinfected mothers may nevertheless manifest morphological changes identifiable by neuroimaging [3]. Furthermore, since the brain is not fully developed even at birth, more subtle neurological damage may only become evident later in delayed developmental milestones. As more children are born in areas where ZIKV epidemics have occurred, and diagnostic tests become better able to differentiate ZIKV from other flavivirus infections, especially dengue, a more complete clinical picture of the outcomes associated with congenital ZIKV infection will undoubtedly emerge. How the fetus is infected by ZIKV is fundamental to informing interventional approaches. Studies in experimental animals confirm that infection during the early stages of pregnancy is more likely to cause the most severe forms of the congenital disease [32_TD$IF][4]. However, it is clear that infection at later stages may also cause fetal disease [32_TD$IF][4,2]. In endemic areas, most pregnant women become infected from the bite of an infected
mosquito. Vaginal inoculation through sexual contact with an infected male is clearly an important form of transmission in nonendemic areas, but its relevance in endemic regions remains unclear [3_TD$IF][5]. While antibodies from previous dengue infections may cause enhancement of ZIKV infection in vitro, the role of previous infections in causing congenital disease remains to be determined. The latter has often been raised to explain the apparent greater frequency of severe congenital Zika syndrome in the Northeast of Brazil. On the other hand, one study in Brazil has shown that areas with lower yellow fever vaccination coverage were more likely to have cases of microcephaly [34_TD$IF][6]. Thus, more detailed studies are needed to determine the relevance of previous and/or concomitant arboviral infections or vaccinations to ZIKV-associated congenital syndromes. Several studies have now evaluated pathological changes in the placenta associated with ZIKV infection in the fetus. Overall, it is clear that maternal ZIKV infection during pregnancy causes placental infection, with significant levels of virus in placental macrophages (Hofbauer cells) and placental villous fibroblasts [35_TD$IF][7,8]. In addition, most studies have shown that fetuses with significant damage have viral RNA or protein in the brain. Therefore, although local placental changes may contribute to fetal disease pathogenesis, it seems that the virus must reach the fetal brain for ensuing central nervous system damage to occur. How does ZIKV reach the developing fetal brain? Virus in the maternal blood must be transmitted across the placenta, in particular, the syncytiotrophoblast. This first barrier is syncytial, capable of producing type I and III interferons. These cells are highly resistant to infection, at least at the time of parturition in humans [36_TD$IF][9], although it is unclear if the levels of type III interferon required to maintain this resistance (which would also likely protect local placental cells from infection) are sustained from
Trends in Microbiology, September 2017, Vol. 25, No. 9
701
early in pregnancy. A recent study has shown that cells from human term placentas, which resist infection, expressed genes associated with antiviral defense but not genes that encode attachment factors linked to ZIKV entry. In contrast, trophoblasts derived from embryonic stem cells lacked strong innate antiviral responses and also expressed attachment factors for ZIKV entry [37_TD$IF][10]. AXL and TIM1, putative receptors for ZIKV, are expressed by cells throughout the placenta and amniotic epithelium, including Hofbauer cells, making these cells susceptible to ZIKV infection in vitro [38_TD$IF][8]. These motile placental macrophages may thus function as Trojan horses, spreading the virus to the rest of the placenta, which in turn may facilitate transmission into the fetal circulation. Viral replication in placental macrophages induced type I IFN, proinflammatory cytokines, and antiviral gene expression, with relatively little cell death [39_TD$IF][7]. However, high levels of pro-inflammatory cytokine expression secondary to infection can affect fetal development, as emphasized by recent findings that cell damage in the developing brain, including premature maturation and apoptosis, is mediated by cytokine outputs from nearby ZIKV-infected cells [40_TD$IF][11]. Experimentally, mice show an early ZIKV-susceptible teratogenic window between gastrulation and organogenesis, suggesting infection of the embryo prior to full placentation. Infection later in gestation resulted in morphologically normal fetuses in about half of the conceptuses and containment of ZIKV within the placenta. However, where fetuses were abnormal, significant placental vascular disruption was also evident, suggesting that impaired materno-fetal circulation also contributes to pathology [32_TD$IF][4]. Unlike most other mammals, mice have a hemochorial placenta, similar to humans. Notwithstanding several anatomical
702
differences – for example, mice have three trophoblast layers (hemotrichorial) separating maternal and fetal circulations, compared to just one ultimately (hemomonochorial) in humans – the direct contact of the trophoblast with the maternal blood circulation in both species makes the mouse a useful experimental approximation of human placental virus transmission. Once in the fetus, and subsequently in the developing fetal brain, ZIKV infection corresponds [41_TD$IF]to the expression of AXL on neural progenitors and glial cells [42_TD$IF][12], although Retallack et al. reported [43_TD$IF]the relative resistance of mature neurons to infection and the susceptibility of glial cells in the same organotypic cultures [4_TD$IF][13]. This is the reverse of the in vivo situation for neurotropic flaviviruses, which tend to infect only neurons in adult humans and animal models. However, the early developmental stage of microglia in the embryo, making [45_TD$IF]these cells more susceptible to productive infection (rather than resistant, like adult microglia), the access by ZIKV to the brain parenchyma provided by the immaturity of the blood–brain barrier, and the ineffectual virus control by an immature fetal adaptive immune response may together permit a more generalized infection of the brain parenchyma in the embryo than in the adult. How the altered maternal immune response during pregnancy contributes to disease during ZIKV infection is an important area for further investigation. Identifying immune markers associated with development of (or protection from) microcephaly and other central nervous system defects will be valuable in determining the key immune processes during infection. Some of these outcomes will be difficult or time-consuming to achieve in human patient populations [46_TD$IF]and we
Trends in Microbiology, September 2017, Vol. 25, No. 9
expect that insights from pregnant animal models of ZIKV infection will make major contributions to our understanding of disease in the next few years. 1 Discipline of Pathology, Bosch Institute, School of Medical Sciences, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School, Charles Perkins Centre, University of Sydney, Camperdown, NSW, Australia 2 Instituto de Ciencias Biologicas, Universidade Federal de
Minas Gerais, Belo Horizonte, Minas Gerais, Brazil 3 Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland 4222, Australia *Correspondence: s.mahalingam@griffith.edu.au (S. Mahalingam). http://dx.doi.org/10.1016/j.tim.2017.05.005 References 1. Kourtis, A.P. et al. (2014) Pregnancy and infection. N. Engl. J. Med. 370, 2211–2218 2. Melo, A.S. et al. (2016) Congenital Zika virus infection: beyond neonatal microcephaly. JAMA Neurol. 73, 1407–1416 3. van der Linden, V. et al. (2016) Description of 13 infants born during October 2015–January 2016 with congenital Zika virus infection without microcephaly at birth – Brazil. MMWR. Morb. Mortal. Wkly Rep. 65, 1343–1348 4. Xavier-Neto, J. (2017) Hydrocephalus and arthrogryposis in an immunocompetent mouse model of ZIKA teratogeny: A developmental study. PLoS Negl. Trop. Dis. e0005363 5. Mansuy, J.M. et al. (2016) Zika virus: high infectious viral load in semen, a new sexually tansmitted pathogen? Lancet Infect. Dis. 16, 405 6. De Góes Cavalcanti, L.P. et al. (2016) Zika virus infection, associated microcephaly, and low yellow fever vaccination coverage in Brazil: is there any causal link? J. Infect. Dev. Ctries. 10, 563–566 7. Quicke, K.M. et al. (2016) Zika virus infects human placental macrophages. Cell Host Microbe 20, 83–90 8. Tabata, T. et al. (2016) Zika virus targets different primary human placental cells, suggesting two routes for vertical transmission. Cell Host Microbe 20, 155–166 9. Bayer, A. et al. (2016) Type III interferons produced by human placental trophoblasts confer protection against Zika virus infection. Cell Host Microbe 19, 705–712 10. Sheridan, M.A. et al. (2017) Vulnerability of primitive human placental trophoblast to Zika virus. Proc. Natl. Acad. Sci. U. S. A. 114, E1587–E1596 11. Bayless, N.L. et al. (2016) Zika virus infection induces cranial neural crest cells to produce cytokines at levels detrimental for neurogenesis. Cell Host Microbe 20, 423– 428 12. Nowakowski, T.J. et al. (2016) Expression analysis highlights AXL as a candidate Zika virus entry receptor in neural stem sells. Cell Stem Cell 18, 591–596 13. Retallack, H. et al. (2016) Zika virus cell tropism in the developing human brain and inhibition by azithromycin. Proc. Natl. Acad. Sci. U. S. A. 113, 14408–14413
