Influenza A virus infection disrupts the function of syncytiotrophoblast cells and contributes to adverse pregnancy outcomes.

Pregnancy heightens susceptibility to influenza A virus (IAV) infection, thereby increasing the risk of severe pneumonia and maternal mortality. It also raises the chances of adverse outcomes in offspring, such as fetal growth restriction, preterm birth, miscarriage, and stillbirth in offsprings. However, the underlying mechanisms behind these effects remain largely unknown. Syncytiotrophoblast cells, crucial in forming the placental barrier, nutrient exchange and hormone secretion, have not been extensively studied for their responses to IAV. In our experiment, we used Forskolin-treated BeWo cells to mimic syncytiotrophoblast cells in vitro, and infected them with H1N1, H5N1 and H7N9 virus stains. Our results showed that syncytiotrophoblast cells, with their higher intensity of sialic acid receptors, strongly support IAV infection and replication. Notably, high-dose viral infection and prolonged exposure resulted in a significant decrease in fusion index, as well as gene and protein expression levels associated with trophoblast differentiation, ß-human chorionic gonadotropin secretion, estrogen and progesterone biosynthesis, and nutrient transport. In pregnant BALB/c mice infected with the H1N1 virus, we observed significant decreases in trophoblast differentiation and hormone secretion gene expression levels. IAV infection also resulted in preterm labor, fetal growth restriction, and increased maternal and fetal morbidity and mortality. Our findings indicate that IAV infection in syncytiotrophoblastic cells can result in adverse pregnancy outcomes by altering trophoblast differentiation, suppressing of ß-hCG secretion, and disrupting placental barrier function.

Placental SARS-CoV-2 distribution correlates with level of tissue oxygenation in COVID-19-associated necrotizing histiocytic intervillositis/perivillous fibrin deposition.

INTRODUCTION: Recent evidence supports the - rare - occurrence of vertical transplacental SARS-CoV-2 transmission. We previously determined that placental expression of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, and associated viral cell entry regulators is upregulated by hypoxia. In the present study, we utilized a clinically relevant model of SARS-CoV-2-associated chronic histiocytic intervillositis/massive perivillous fibrin deposition (CHIV/MPFVD) to test the hypothesis that placental hypoxia may facilitate placental SARS-CoV-2 infection. METHODS: We performed a comparative immunohistochemical and/or RNAscope in-situ hybridization analysis of carbonic anhydrase IX (CAIX, hypoxia marker), ACE2 and SARS-CoV-2 expression in free-floating versus fibrin-encased chorionic villi in a 20-weeks' gestation placenta with SARS-CoV-2-associated CHIV/MPVFD. RESULTS: The levels of CAIX and ACE2 immunoreactivity were significantly higher in trophoblastic cells of fibrin-encased villi than in those of free-floating villi, consistent with hypoxia-induced ACE2 upregulation. SARS-CoV-2 showed a similar preferential localization to trophoblastic cells of fibrin-encased villi. DISCUSSION: The localization of SARS-CoV-2 to hypoxic, fibrin-encased villi in this placenta with CHIV/MPVFD suggests placental infection and, therefore, transplacental SARS-CoV-2 transmission may be promoted by hypoxic conditions, mediated by ACE2 and similar hypoxia-sensitive viral cell entry mechanisms. Understanding of a causative link between placental hypoxia and SARS-CoV-2 transmittability may potentially lead to the development of alternative strategies for prevention of intrauterine COVID-19 transmission.

Murine Trophoblast Stem Cells and Their Differentiated Cells Attenuate Zika Virus In Vitro by Reducing Glycosylation of the Viral Envelope Protein.

Zika virus (ZIKV) infection during pregnancy can cause devastating fetal neuropathological abnormalities, including microcephaly. Most studies of ZIKV infection in pregnancy have focused on post-implantation stage embryos. Currently, we have limited knowledge about how a pre-implantation stage embryo deals with a viral infection. This study investigates ZIKV infection on mouse trophoblast stem cells (TSCs) and their in vitro differentiated TSCs (DTSCs), which resemble the cellular components of the trophectoderm layer of the blastocyst that later develops into the placenta. We demonstrate that TSCs and DTSCs are permissive to ZIKV infection; however, ZIKV propagated in TSCs and DTSCs exhibit substantially lower infectivity, as shown in vitro and in a mouse model compared to ZIKV that was generated in Vero cells or mouse embryonic fibroblasts (MEFs). We further show that the low infectivity of ZIKV propagated in TSCs and DTSCs is associated with a reduced level of glycosylation on the viral envelope (E) proteins, which are essential for ZIKV to establish initial attachment by binding to cell surface glycosaminoglycans (GAGs). The decreased level of glycosylation on ZIKV E is, at least, partially due to the low-level expression of a glycosylation-related gene, Hexa, in TSCs and DTSCs. Furthermore, this finding is not limited to ZIKV since similar observations have been made as to the chikungunya virus (CHIKV) and West Nile virus (WNV) propagated in TSCs and DTSCs. In conclusion, our results reveal a novel phenomenon suggesting that murine TSCs and their differentiated cells may have adapted a cellular glycosylation system that can limit viral infectivity by altering the glycosylation of viral envelope proteins, therefore serving as a unique, innate anti-viral mechanism in the pre-implantation stage embryo.

Evidence for HPV DNA in the placenta of women who resorted to elective abortion.

BACKGROUND: It is believed that HPV infection can result in the death of placental trophoblasts and cause miscarriages or preterm birth. In clinical cases of placental villi positive for HPV DNA reported by other authors, contamination is suspected in the act of crossing the cervical canal. We analyzed placental samples of women who resorted to elective abortion obtained by hysterosuction of ovular material, bypassing any contact with the cervical canal and vagina. METHODS: We studied the chorionic villi of the placenta of 64 women who resorted to voluntary termination of pregnancy, in the first trimester. To avoid contamination of the villi by the cervical canal, we analyzed placental samples obtained by hysterosuction of ovular material, bypassing any contact with the cervical canal and vagina. All samples of chorionic villi were manually selected from the aborted material and subjected to research for HPV DNA. RESULTS: HPV DNA was detected in 10 out of 60 women (16.6%). The HPV DNA identified in the placenta belonged to genotypes 6, 16, 35, 53, and 90. CONCLUSION: The study shows that papillomavirus DNA can infect the placenta and that placenta HPV infection can occur as early as the first trimester of pregnancy.

Hofbauer Cells and COVID-19 in Pregnancy.

CONTEXT.­: SARS-CoV-2 can undergo maternal-fetal transmission, heightening interest in the placental pathology findings from this infection. Transplacental SARS-CoV-2 transmission is typically accompanied by chronic histiocytic intervillositis together with necrosis and positivity of syncytiotrophoblast for SARS-CoV-2. Hofbauer cells are placental macrophages that have been involved in viral diseases, including HIV and Zika virus, but their involvement in SARS-CoV-2 is unknown. OBJECTIVE.­: To determine whether SARS-CoV-2 can extend beyond the syncytiotrophoblast to enter Hofbauer cells, endothelium, and other villous stromal cells in infected placentas of liveborn and stillborn infants. DESIGN.­: Case-based retrospective analysis by 29 perinatal and molecular pathology specialists of placental findings from a preselected cohort of 22 SARS-CoV-2-infected placentas delivered to pregnant women testing positive for SARS-CoV-2 from 7 countries. Molecular pathology methods were used to investigate viral involvement of Hofbauer cells, villous capillary endothelium, syncytiotrophoblast, and other fetal-derived cells. RESULTS.­: Chronic histiocytic intervillositis and trophoblast necrosis were present in all 22 placentas (100%). SARS-CoV-2 was identified in Hofbauer cells from 4 of 22 placentas (18.2%). Villous capillary endothelial staining was positive in 2 of 22 cases (9.1%), both of which also had viral positivity in Hofbauer cells. Syncytiotrophoblast staining occurred in 21 of 22 placentas (95.5%). Hofbauer cell hyperplasia was present in 3 of 22 placentas (13.6%). In the 7 cases having documented transplacental infection of the fetus, 2 (28.6%) occurred in placentas with Hofbauer cell staining positive for SARS-CoV-2. CONCLUSIONS.­: SARS-CoV-2 can extend beyond the trophoblast into the villous stroma, involving Hofbauer cells and capillary endothelial cells, in a small number of infected placentas. Most cases of SARS-CoV-2 transplacental fetal infection occur without Hofbauer cell involvement.

Zika virus infection of first trimester trophoblast cells affects cell migration, metabolism and immune homeostasis control.

Zika virus (ZIKV) re-emerged after circulating almost undetected for many years and the last spread in 2015 was the major outbreak reported. ZIKV infection was associated with congenital fetal growth anomalies such as microcephaly, brain calcifications, and low birth weight related to fetal growth restriction. In this study, we investigated the effect of ZIKV infection on first trimester trophoblast cell function and metabolism. We also studied the interaction of trophoblast cells with decidual immune populations. Results presented here demonstrate that ZIKV infection triggered a strong antiviral response in first trimester cytotrophoblast-derived cells, impaired cell migration, increased glucose uptake and GLUT3 expression, and reduced brain derived neurotrophic factor (BDNF) expression. ZIKV infection also conditioned trophoblast cells to favor a tolerogenic response since an increased recruitment of CD14+ monocytes bearing an anti-inflammatory profile, increased CD4+ T cells and NK CD56Dim and NK CD56Bright populations and an increment in the population CD4+ FOXP3+ IL-10+ cells was observed. Interestingly, when ZIKV infection of trophoblast cells occurred in the presence of the vasoactive intestinal peptide (VIP) there was lower detection of viral RNA and reduced toll-like receptor-3 and viperin messenger RNA expression, along with reduced CD56Dim cells trafficking to trophoblast conditioned media. The effects of ZIKV infection on trophoblast cell function and immune-trophoblast interaction shown here could contribute to defective placentation and ZIKV persistence at the fetal-maternal interface. The inhibitory effect of VIP on ZIKV infection of trophoblast cells highlights its potential as a candidate molecule to interfere ZIKV infection during early pregnancy.

The Role of Congenital Cytomegalovirus Infection in Adverse Birth Outcomes: A Review of the Potential Mechanisms.

Human cytomegalovirus (CMV) is a major cause of nonhereditary adverse birth outcomes, including hearing and visual loss, neurologic deficits, and intrauterine growth retardation (IUGR), and may contribute to outcomes such as stillbirth and preterm delivery. However, the mechanisms by which CMV could cause adverse birth outcomes are not fully understood. This study reviewed proposed mechanisms underlying the role of CMV in stillbirth, preterm birth, and IUGR. Targeted literature searches were performed in PubMed and Embase to identify relevant articles. Several potential mechanisms were identified from in vitro studies in which laboratory-adapted and low-passage strains of CMV and various human placental models were used. Potential mechanisms identified included impairment of trophoblast progenitor stem cell differentiation and function, impairment of extravillous trophoblast invasiveness, dysregulation of Wnt signaling pathways in cytotrophoblasts, tumor necrosis factor-α mediated apoptosis of trophoblasts, CMV-induced cytokine changes in the placenta, inhibition of indoleamine 2,3-dioxygenase activity, and downregulation of trophoblast class I major histocompatibility complex molecules. Inherent challenges for the field remain in the identification of suitable in vivo animal models. Nonetheless, we believe that our review provides useful insights into the mechanisms by which CMV impairs placental development and function and how these changes could result in adverse birth outcomes.

HTLV-1 targets human placental trophoblasts in seropositive pregnant women.

Human T cell leukemia virus type 1 (HTLV-1) is mainly transmitted vertically through breast milk. The rate of mother-to-child transmission (MTCT) through formula feeding, although significantly lower than through breastfeeding, is approximately 2.4%-3.6%, suggesting the possibility of alternative transmission routes. MTCT of HTLV-1 might occur through the uterus, birth canal, or placental tissues; the latter is known as transplacental transmission. Here, we found that HTLV-1 proviral DNA was present in the placental villous tissues of the fetuses of nearly half of pregnant carriers and in a small number of cord blood samples. An RNA ISH assay showed that HTLV-1-expressing cells were present in nearly all subjects with HTLV-1-positive placental villous tissues, and their frequency was significantly higher in subjects with HTLV-1-positive cord blood samples. Furthermore, placental villous trophoblasts expressed HTLV-1 receptors and showed increased susceptibility to HTLV-1 infection. In addition, HTLV-1-infected trophoblasts expressed high levels of viral antigens and promoted the de novo infection of target T cells in a humanized mouse model. In summary, during pregnancy of HTLV-1 carriers, HTLV-1 was highly expressed in placental villous tissues, and villous trophoblasts showed high HTLV-1 sensitivity, suggesting that MTCT of HTLV-1 occurs through the placenta.

Placental Alkaline Phosphatase Promotes Zika Virus Replication by Stabilizing Viral Proteins through BIP.

Zika virus (ZIKV) infection during pregnancy causes intrauterine growth defects and microcephaly, but knowledge of the mechanism through which ZIKV infects and replicates in the placenta remains elusive. Here, we found that ALPP, an alkaline phosphatase expressed primarily in placental tissue, promoted ZIKV infection in both human placental trophoblasts and astrocytoma cells. ALPP bound to ZIKV structural and nonstructural proteins and thereby prevented their proteasome-mediated degradation and enhanced viral RNA replication and virion biogenesis. In addition, the function of ALPP in ZIKV infection depends on its phosphatase activity. Furthermore, we demonstrated that ALPP was stabilized through interactions with BIP, which is the endoplasmic reticulum (ER)-resident heat shock protein 70 chaperone. The chaperone activity of BIP promoted ZIKV infection and mediated the interaction between ALPP and ZIKV proteins. Collectively, our findings reveal a previously unrecognized mechanism through which ALPP facilitates ZIKV replication by coordinating with the BIP protein.IMPORTANCE ZIKV is a recently emerged mosquito-borne flavivirus that can cause devastating congenital Zika syndrome in pregnant women and Guillain-Barré syndrome in adults, but how ZIKV specifically targets the placenta is not well understood. Here, we identified an alkaline phosphatase (ALPP) that is expressed primarily in placental tissue and promotes ZIKV infection by colocalizing with ZIKV proteins and preventing their proteasome-mediated degradation. The phosphatase activity of ALPP could be required for optimal ZIKV infection, and ALPP is stabilized by BIP via its chaperone activity. This report provides novel insights into host factors required for ZIKV infection, which potentially has implications for ZIKV infection of the placenta.

Effects of cytomegalovirus infection on extravillous trophoblast cells invasion and immune function of NK cells at the maternal-fetal interface.

Cytomegalovirus (CMV) is one of the most common intrauterine infection virus, which can cause intrauterine transmission through the placenta, resulting in abortion, stillbirth and congenital malformations. In this study, the co-culture extravillous trophoblast (EVT) HTR8/SVneo cell model of CMV infection was established in vitro. The toxicity of CMV infected EVT was determined, and then, the cell invasion experiment was conducted to evaluate the effect on the invasion ability of EVT cell lines. Western blot and real-time PCR were used to detect the related cytokines in the PI3K/AKT signalling pathway in cells. Flow cytometry was used to detect the immune function related factors of the supernatant of CMV culture on decidual NK cells. The TCID50 of CMV virus was 10-5.4 . The results of immunofluorescence showed that a large number of fluorescent green of CMV pp65 antigen signals appeared in the cytoplasm of CMV infection group. CMV could infect and replicate EVT cells and inhibited cell proliferation. The expression of proteins PDK1, AKT-S473 and AKT-S308 was significantly increased in CMV infection group. The levels of IL-17, IL-4 and IFN-γ were 8.7 ± 0.48%, 12.17 ± 0.61% and 6.66 ± 0.25%, respectively, in CMV infection group. The above results indicated that CMV infection inhibited EVT cells proliferation, weakened the invasion ability and inhibited the immune function of NK cells at the maternal-fetal interface, resulting in the abnormal maternal-fetal crosstalk.

Zika Virus Targets Multiple Tissues and Cell Types During the First Trimester of Pregnancy.

The 2016 Zika virus (ZIKV) outbreak in the Americas has been characterized by an increased association frequency of fetal neuropathological abnormalities. To have a comprehensive and accurate knowledge of key elements of the clinically observed neurologic dysfunctions in Zika-infected babies, ZIKV transmission from mother to fetus needs to be deeply studied. Thus, it is important to determine the role of both virus-targeted cells and tissues within the mother-fetus interface. Cellular tropism and mechanisms of ZIKV transmission from mother to the fetus during early pregnancy still remain unknown on many aspects. To improve the characterization of the maternal-fetal ZIKV transmission, we have set up an ex vivo model using an organ culture approach with a light-invasive sampling from the first trimester of pregnancy samples. Thus, here we provide evidence that circulating epidemic ZIKV strains from Latin America widely target and destroy reproductive tissues, including the decidua basalis, fetal placenta, and umbilical cord. In addition, we show that ZIKV is able to differentially replicate in a large range of both maternal and fetal cells, including decidual fibroblasts and macrophages, fetal trophoblast and Hofbauer cells, as well as umbilical cord mesenchymal stem cells. This primary and broad ZIKV cellular tropism and the resulting abundant cytopathic-induced tissue effects during the first trimester of pregnancy show the upstream path of clinically observed congenital damages.

Endogenous Retroviral Envelope Syncytin Induces HIV-1 Spreading and Establishes HIV Reservoirs in Placenta.

Radical cure of HIV-1 (HIV) is hampered by the establishment of HIV reservoirs and persistent infection in deep tissues despite suppressive antiretroviral therapy (ART). Here, we show that among HIV-positive women receiving suppressive ART, cells from placental tissues including trophoblasts contain HIV RNA and DNA. These viruses can be reactivated by latency reversal agents. We find that syncytin, the envelope glycoprotein of human endogenous retrovirus family W1 expressed on placental trophoblasts, triggers cell fusion with HIV-infected T cells. This results in cell-to-cell spread of HIV to placental trophoblasts. Such cell-to-cell spread of HIV is less sensitive to ART than free virus. Replication in syncytin-expressing cells can also produce syncytin-pseudotyped HIV, further expanding its ability to infect non-CD4 cells. These previously unrecognized mechanisms of HIV entry enable the virus to bypass receptor restriction to infect host barrier cells, thereby facilitating viral transmission and persistent infection in deep tissues.

Villitis of unknown etiology and chronic deciduitis are not associated with human papilloma virus and enterovirus infection.

Villitis of unknown etiology (VUE) and chronic deciduitis with plasma cells (CD) are supposed to be non infectious placental lesions caused by a pathologic immune reaction similar to a host versus graft mechanism. In some investigations, infection of human trophoblastic cells with human papilloma virus (HPV) has been described, and a relationship with miscarriage, preeclampsia, and chronic inflammatory placental lesions has been suspected. Infection with enterovirus, especially Coxsackievirus, has been observed in cases with spontaneous abortion and adverse perinatal outcome, respectively. We investigated 20 cases with VUE and 30 cases with chronic deciduitis with plasma cells. The placenta specimens were analyzed for expression of HPV capsid protein by immunohistochemistry, for presence of HPV DNA via polymerase chain reaction (PCR), and for presence of enterovirus mRNA using RT-PCR, respectively. VUE was associated with maternal diseases: atopic lesions in 21%, other autoimmune diseases in 15.5%, and obesity in 31.5%, respectively. Birth weight below the 10th percentile was detected in 63% of the cases with VUE. Chronic deciduitis was associated with preterm labor and preterm premature rupture of membranes (26%). Intrauterine fetal demise occurred in 5 cases with CD (18.5%). HPV DNA, HPV capsid protein, and enterovirus mRNA were not detected in all investigated VUE or CD cases. Our investigations show that a causal role for enterovirus and human papilloma virus in the development of VUE and CD is unlikely. Therefore, HPV vaccination is unlikely to reduce the incidence of VUE and CD in the future.

HHV-6A Infection of Endometrial Epithelial Cells Affects miRNA Expression and Trophoblast Cell Attachment.

We recently reported that human herpesvirus 6 (HHV-6) infection is frequently present in endometrial tissue of women with unexplained infertility, and that virus infection induces a profound remodulation of miRNA expression in human cells of different origin. Since specific miRNA patterns have been associated with specific pregnancy outcomes, we aimed to analyze the impact of HHV-6A infection on miRNAs expression and trophoblast receptivity in human endometrial cells. To this purpose, a human endometrial cell line (HEC-1A) was infected with HHV-6A and analyzed for alterations in the expression of miRNAs and for permissiveness to the attachment of a human choriocarcinoma trophoblast cell line (JEG-3). The results showed that HHV-6A infection of endometrial cells up-modulates miR22 (26-fold), miR15 (19.5-fold), and miR196-5p (12.1 fold), that are correlated with implant failure, and down-modulates miR18 (11.4 fold), miR101-3p (4.6 fold), miR181-5p (4.9 fold), miR92 (3.3 fold), and miR1207-5p (3.9 fold), characterized by a low expression in preeclampsia. Moreover, HHV-6A-infected endometrial cells infected resulted less permissive to the attachment of trophoblast cells. In conclusion, collected data suggest that HHV-6A infection could modify miRNA expression pattern and control of trophoblast cell adhesion of endometrial cells, undermining a correct trophoblast cell attachment on endometrial cells.

Differential Expression of PDGF Receptor-α in Human Placental Trophoblasts Leads to Different Entry Pathways by Human Cytomegalovirus Strains.

Human cytomegalovirus (CMV) is the leading non-genetic cause of fetal malformation in developed countries. CMV placental infection is a pre-requisite for materno-fetal transmission of virus, and fetal infection. We investigated the roles of the viral pentameric complex gH/gL/pUL128-pUL131A, and cellular platelet-derived growth factor receptor-α (PDGFRα) for CMV infection in first trimester extravillous-derived (SGHPL-4) and villous-derived (HTR-8/SVneo) trophoblast cells. Infection with four CMV clinical and laboratory strains (Merlin, TB40E, Towne, AD169), and Merlin deletion mutants of UL128-, UL130-, and UL131A-genes, showed a cell type-dependent requirement of the viral pentameric complex for infection of trophoblast cells. The viral pentameric complex was essential for infection of villous trophoblasts, but non-essential for extravillous trophoblasts. Blocking of PDGFRα in extravillous trophoblasts, which naturally express PDGFRα, inhibited entry of pentameric complex-deficient CMV strains, but not the entry of pentameric positive CMV strains. Transient expression of PDGFRα in villous trophoblasts, which are naturally deficient in PDGFRα, promoted the entry of CMV strains lacking gH/gL/pUL128-pUL131A, but had no effect on entry of pentameric positive CMV strains. These results suggest PDGFRα is an important cell receptor for entry of CMV mutant strains lacking gH/gL/pUL128-pUL131A complexes in some placental cells, suggesting these entry pathways could be potential antiviral targets.

Breast Cancer Resistance Protein (BCRP/ABCG2) Inhibits Extra Villous Trophoblast Migration: The Impact of Bacterial and Viral Infection.

Extravillous trophoblasts (EVT) migration into the decidua is critical for establishing placental perfusion and when dysregulated, may lead to pre-eclampsia (PE) and intrauterine growth restriction (IUGR). The breast cancer resistance protein (BCRP; encoded by ABCG2) regulates the fusion of cytotrophoblasts into syncytiotrophoblasts and protects the fetus from maternally derived xenobiotics. Information about BCRP function in EVTs is limited, however placental exposure to bacterial/viral infection leads to BCRP downregulation in syncitiotrophoblasts. We hypothesized that BCRP is involved in the regulation of EVT function and is modulated by infection/inflammation. We report that besides syncitiotrophoblasts and cytotrophoblasts, BCRP is also expressed in EVTs. BCRP inhibits EVT cell migration in HTR8/SVneo (human EVT-like) cells and in human EVT explant cultures, while not affecting cell proliferation. We have also shown that bacterial-lipopolysaccharide (LPS)-and viral antigens-single stranded RNA (ssRNA)-have a profound effect in downregulating ABCG2 and BCRP levels, whilst simultaneously increasing the migration potential of EVT-like cells. Our study reports a novel function of BCRP in early placentation and suggests that exposure of EVTs to maternal infection/inflammation could disrupt their migration potential via the downregulation of BCRP. This could negatively influence placental development/function, contribute to existing obstetric pathologies, and negatively impact pregnancy outcomes and maternal/neonatal health.

CD46 facilitates entry and dissemination of human cytomegalovirus.

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.

African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast.

Zika virus (ZIKV) drew worldwide attention when a recent epidemic was linked to fetal microcephaly. Here we used human embryonic stem cell derived trophoblasts as a model for primitive placental trophoblast to test the hypothesis that there are differences in how the two genetically distinct ZIKV lineages, African (AF) and Asian (AS), target the human placenta. Upon infection with three AF (ib-H30656, SEN/1984/41525-DAK, and MR-766) and three AS (FSS13025, MexI-44, and PANcdc259249) ZIKV strains, we observed that severe placental cell lysis was only induced after infection with AF strains, while viral replication rates remained similar between both lineages. Differences in cytopathic effects (CPE) were not observed in Vero cells, indicating that the AF strains were not inherently superior at cell lysis. Taken together, we propose that infection with AF strains of ZIKV early in pregnancy would likely result in pregnancy loss, rather than allow further fetal development with accompanying brain damage. Our results also suggest that the long term laboratory-adapted MR-766 strain does not behave aberrantly in cell culture relative to other AF lineage strains.

Chromosome 19 microRNAs exert antiviral activity independent from type III interferon signaling.

INTRODUCTION: Cultured primary human trophoblasts (PHT), derived from term placentas, are relatively resistant to infection by diverse viruses. The resistance can be conferred to non-trophoblastic cells by pre-exposing them to medium that was conditioned by PHT cells. This antiviral effect is mediated, at least in part, by microRNAs (miRNA) expressed from the chromosome 19 microRNA cluster (C19MC). Recently we showed that PHT cells and cells pre-exposed to PHT medium are also resistant to infection by Zika virus (ZIKV), an effect mediated by the constitutive release of the type III interferons (IFN) IFN lambda-1 and IFN lambda-2 in trophoblastic medium. We hypothesized that trophoblastic C19MC miRNA are active against ZIKV, and assessed the interaction of this pathway with IFN lambda-1 - mediated resistance. METHODS: Term PHT cells were cultured using standard techniques. An osteosarcoma cell line (U2OS) was used as non-trophoblastic cells, which were infected with either ZIKV or vesicular stomatitis virus (VSV). Trophoblastic extracellular vesicles (EVs) were produced by gradient ultracentrifugation. RT-qPCR was used to determine viral infection, cellular or medium miRNA levels and the expression of interferon-stimulated genes. RESULTS: We showed that C19MC miRNA attenuate infection of U2OS cells by ZIKV, and that C19MC miRNA or exosomes that contain C19MC miRNA did not influence the type III IFN pathway. Similarly, cell exposure to recombinant IFN lambda-1 had no effect on miRNA expression, and these pathways did not exhibit synergistic interaction. DISCUSSION: PHT cells exert antiviral activity by at least two independent mechanisms, mediated by C19MC miRNA and by type III IFNs.

Human papillomavirus infects placental trophoblast and Hofbauer cells, but appears not to play a causal role in miscarriage and preterm labor.

INTRODUCTION: Recently, an association between human papillomavirus infection and both spontaneous abortion and spontaneous preterm delivery was suggested. However, the reported human papillomavirus prevalence in pregnant women varies considerably and reliable conclusions are difficult. We aimed to investigate human papillomavirus infection in placental tissue of a Danish study cohort. Furthermore, we studied the cellular localization of human papillomavirus. MATERIAL AND METHODS: In this prospective case-control study, placental tissue was analyzed for human papillomavirus infection by nested PCR in the following four study groups: full-term delivery (n = 103), spontaneous preterm delivery (n = 69), elective abortion (n = 54), and spontaneous abortion (n = 44). Moreover, human papillomavirus cellular target was identified using in situ hybridization. RESULTS: Human papillomavirus prevalence in placental tissue was 8.7% in full-term deliveries, 8.8% in spontaneous preterm deliveries, 10.9% in spontaneous abortions, and 20.4% in elective abortions. Twelve different human papillomavirus types were detected, and placental human papillomavirus infection was associated to a disease history of cervical cancer. Human papillomavirus DNA was identified in trophoblast cells, cells of the placental villi mesenchyme including Hofbauer cells, and in parts of the encasing endometrium. CONCLUSION: Placental human papillomavirus infections are not likely to constitute a risk factor for spontaneous preterm labor or spontaneous abortions in the Danish population, although an effect of human papillomavirus DNA in placental cells cannot be excluded.