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A tool to probe SARS-CoV-2 biology To develop therapies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emerging variants, it is important to understand the viral biology and the effect of mutations. However, this is challenging because live virus can only be studied in a few laboratories that meet stringent safety standards. Syed et al . describe a virus-like particle (VLP) that comprises the four SARS-CoV-2 structural proteins, but instead of packaging viral RNA, it packages messenger RNA (mRNA) that expresses a reporter protein (see the Perspective by Johnson and Menachery). The amount of reporter expressed in receiver cells depends on the efficiency of packaging and assembly in the producer cells and the efficiency of entry into receiver cells. Mutations in the nucleocapsid protein that are found in more transmissible variants increase mRNA packaging and expression. The VLPs provide a platform for studying the effect of mutations in the structural proteins and for screening therapeutics. —VV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Inhibitors of bromodomain and extraterminal domain (BET) proteins are possible anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here we show that BET proteins should not be inactivated therapeutically because theyare critical antiviral factors at the post-entry level. Depletion of BRD3 or BRD4 in cells overexpressing ACE2 exacerbates SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon productioninduced by SARS-CoV-2, a process phenocopied by the envelope (E)protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain ofBRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responsesvia BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here we developed covalent nanobodies capable of binding with SARS-CoV-2 spike protein irreversibly via proximity-enabled reactive therapeutic (PERx) mechanism. A novel latent bioreactive amino acid FFY was designed and genetically encoded into nanobodies to accelerate PERx reaction rate. After covalent engineering, nanobodies binding with the Spike in the down state, but not in the up state, were discovered to possess striking enhancement in inhibiting viral infection. In comparison with the noncovalent wildtype nanobody, the FFY-incorporated covalent nanobody neutralized both authentic SARS-CoV-2 and its Alpha and Delta variants with potency drastically increased over tens of folds. This PERx-enabled covalent nanobody strategy and uncovered insights on potency increase can be valuable to developing effective therapeutics for various viral infections.

The Omicron SARS-CoV-2 virus contains extensive sequence changes relative to the earlier arising B.1, B.1.1 and Delta SARS-CoV-2 variants that have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (SC2-VLPs), we examined mutations in all four structural proteins and found that Omicron showed 3-fold higher capsid assembly and cell entry relative to Delta, a property conferred by S and N protein mutations. Thirty-eight antisera samples from individuals vaccinated with Pfizer/BioNTech, Moderna, Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had 15-fold lower efficacy to prevent cell transduction by VLPs containing the Omicron mutations relative to the ancestral B.1 spike protein. A third dose of Pfizer vaccine elicited substantially higher neutralization titers against Omicron, resulting in detectable neutralizing antibodies in 8 out of 8 subjects compared to 1 out of 8 pre-boost. Furthermore, the monoclonal antibody therapeutics Casirivimab and Imdevimab had robust neutralization activity against B.1, B.1.1 or Delta VLPs but no detectable neutralization of Omicron VLPs. Our results suggest that Omicron is more efficient at assembly and cell entry compared to Delta, and the antibody response triggered by existing vaccines or previous infection, at least prior to boost, will have limited ability to neutralize Omicron. In addition, some currently available monoclonal antibodies will not be useful in treating Omicron-infected patients., One-Sentence Summary: Omicron SARS-CoV-2 virus-like particles have enhanced infectivity that is only weakly neutralized by vaccination without boost or prior infection, or antibody therapeutics.

Newly evolved SARS-CoV-2 variants are driving ongoing outbreaks of COVID-19 around the world. Efforts to determine why these viral variants have improved fitness are limited to mutations in the viral spike (S) protein and viral entry steps using non-SARS-CoV-2 viral particles engineered to display S. Here we show that SARS-CoV-2 virus-like particles can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and rapid dissection of multiple steps in the viral life cycle. Identification of an RNA packaging sequence was critical for engineered transcripts to assemble together with SARS-CoV-2 structural proteins S, nucleocapsid (N), membrane (M) and envelope (E) into non-replicative SARS-CoV-2 virus-like particles (SC2-VLPs) that deliver these transcripts to ACE2- and TMPRSS2-expressing cells. Using SC2-VLPs, we tested the effect of 30 individual mutations within the S and N proteins on particle assembly and entry. While S mutations unexpectedly did not affect these steps, SC2-VLPs bearing any one of four N mutations found universally in more-transmissible viral variants (P199L, S202R, R203M and R203K) showed increased particle production and up to 10-fold more reporter transcript expression in receiver cells. Our study provides a platform for rapid testing of viral variants outside a biosafety level 3 setting and identifies viral N mutations and viral particle assembly as mechanisms to explain the increased spread of current viral variants, including Delta (N:R203M).One-Sentence SummaryR203M substitution within SARS-CoV-2 N, found in delta variant, improves RNA packaging into virus-like particles by 10-fold.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, yet there are no established treatments for preventing maternal-fetal transmission. During first trimester, HCMV replicates in basal decidua that functions as a reservoir for virus and source of transmission to the attached placenta and fetal hemiallograft but also contains immune cells, including natural killer cells, macrophages, and T cell subsets, that respond to pathogens, protecting the placenta and fetus. However, the specific cellular and cytokine responses to infection are unknown, nor are the immune correlates of protection that guide development of therapeutic strategies. Here we survey immune cell phenotypes in intact explants of basal decidua infected with a clinical pathogenic HCMV strain ex vivo and identify specific changes occurring in response to infection in the tissue environment. Using 4-color immunofluorescence microscopy, we found that at 3days postinfection, virus replicates in decidual stromal cells and epithelial cells of endometrial glands. Infected cells and effector memory CD8+ T cells (TEM) in contact with them make IFN-γ. CD8+ TEM cells produce granulysin and cluster at sites of infection in decidua and the epithelium of endometrial glands. Quantification indicated expansion of two immune cell subtypes-CD8+ TEM cells and, to a lesser extent, iNKT cells. Approximately 20% of immune cells were found in pairs in both control and infected decidua, suggesting frequent cross-talk in the microenvironment of decidua. Our findings indicate a complex immune microenvironment in basal decidua and suggest CD8+ TEM cells play a role in early responses to decidual infection in seropositive women.

Background During pregnancy, the Zika flavivirus (ZIKV) infects human placentas, inducing defects in the developing fetus. The flavivirus nonstructural protein 1 (NS1) alters glycosaminoglycans on the endothelium, causing hyperpermeability in vitro and vascular leakage in vivo in a tissue-dependent manner. The contribution of ZIKV NS1 to placental dysfunction during ZIKV infection remains unknown. Methods We examined the effect of ZIKV NS1 on expression and release of heparan sulfate (HS), hyaluronic acid (HA), and sialic acid on human trophoblast cell lines and anchoring villous explants from first-trimester placentas infected with ZIKV ex vivo. We measured changes in permeability in trophoblasts and stromal cores using a dextran-based fluorescence assay and changes in HA receptor expression using immunofluorescent microscopy. Results ZIKV NS1 in the presence and absence of ZIKV increased the permeability of anchoring villous explants. ZIKV NS1 induced shedding of HA and HS and altered expression of CD44 and lymphatic endothelial cell HA receptor-1, HA receptors on stromal fibroblasts and Hofbauer macrophages in villous cores. Hyaluronidase was also stimulated in NS1-treated trophoblasts. Conclusions These findings suggest that ZIKV NS1 contributes to placental dysfunction via modulation of glycosaminoglycans on trophoblasts and chorionic villi, resulting in increased permeability of human placentas.

Congenital human cytomegalovirus (HCMV) infection is a major viral cause of birth defects, including microcephaly, neurological deficits, loss of hearing and vision, and intrauterine growth restriction. Despite its public health significance, there is no approved treatment for congenital infection during pregnancy; existing antivirals have unacceptable toxicities. The mechanisms of HCMV-induced placental injury, reduced capacity for compensatory development and transmission to the fetus are poorly understood, limiting the development of alternative strategies for clinical management of the disease. Recently, self-renewing, multipotent trophoblast progenitor cells (TBPCs) were reported to reside in the chorion of the human placenta and differentiate into the mature trophoblast subtypes - transport syncytiotrophoblasts and invasive cytotrophoblasts - forming chorionic villi, the functional units of the placenta. HCMV infects TBPCs, reducing the population of progenitor cells and their functional capacity to self-renew, migrate and differentiate. Human TBPCs and chorionic villus explants from first trimester represent relevant models for evaluating efficacies of new antiviral agents in protecting and restoring growth of the developing placenta in response to adverse conditions. Correlating pathology from complications of congenital HCMV infection with impaired development in the tissue environment of anchoring villus explants and defects in TBPC differentiation may enable identification of molecular pathways that could serve as targets for intervention. Here we summarize studies that could open up novel avenues of research on potential therapeutics to sustain placental development, promote differentiation and improve function and pregnancy outcomes.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects worldwide, yet the most effective strategies for preventing virus transmission during pregnancy are unknown. We measured the efficacy of human monoclonal antibodies (mAbs) to HCMV attachment/entry factors glycoprotein B (gB) and the pentameric complex, gH/gL-pUL128&ndash, 131, in preventing infection and spread of a clinical strain in primary placental cells and explants of developing anchoring villi. A total of 109 explants from five first-trimester placentas were cultured, and infection was analyzed in over 400 cell columns containing ~120,000 cytotrophoblasts (CTBs). mAbs to gB and gH/gL, 3-25 and 3-16, respectively, neutralized infection in stromal fibroblasts and trophoblast progenitor cells. mAbs to pUL128-131 of the pentameric complex, 1-103 and 2-18, neutralized infection of amniotic epithelial cells better than mAbs 3-25 and 3-16 and hyperimmune globulin. Select mAbs neutralized infection of cell column CTBs, with mAb 2-18 most effective, followed by mAb 3-25. Treatment of anchoring villi with mAbs postinfection reduced spread in CTBs and impaired formation of virion assembly compartments, with mAb 2-18 achieving better suppression at lower concentrations. These results predict that antibodies generated by HCMV vaccines or used for passive immunization have the potential to reduce transplacental transmission and congenital disease.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via NK cell receptors (NKRs). They serve an important role in controlling viruses and have been described as being particularly important in managing herpesvirus infections. Among the various NKRs, inhibitory NKR-P1 receptors have recently being shown to be targeted by virally-encoded proteins of cytomegalovirus (CMV), a member of the beta-herpesvirus family. In rodents, the NKR-P1B receptor interacts with Clec2d/Clr-b, an inhibitory ligand abundantly expressed on healthy cells. Clr-b is downregulated during stress and serves to alert NK cells of pathogenesis. Interestingly, both rat and mouse CMVs encode viral ligands (RCTL and m12 respectively) that directly engage the NKR-P1B receptor and serve as decoys to subvert NK recognition. In humans, the homologous receptor is NKR-P1A/CD161 and binds CLEC2D/LLT-1. Given the findings in rodents, we are investigating if human CMV (HCMV) similarly employs a mechanism to escape NK detection through NKR-P1A/CD161. Using cell reporters capable of detecting receptor-ligand interactions and various HCMV strains, we are undergoing a systematic approach address this hypothesis.

BACKGROUND: Maternal Zika virus (ZIKV) infection with prolonged viremia leads to fetal infection and congenital Zika syndrome. Previously, we reported that ZIKV infects primary cells from human placentas and fetal membranes. Here, we studied viral replication in numerous explants of anchoring villi and basal decidua from first-trimester human placentas and midgestation amniotic epithelial cells (AmEpCs). METHODS: Explants and AmEpCs were infected with American and African ZIKV strains at low multiplicities, and ZIKV proteins were visualized by immunofluorescence. Titers of infectious progeny, cell proliferation, and invasiveness were quantified. RESULTS: In anchoring villus, ZIKV replicated reproducibly in proliferating cytotrophoblasts in proximal cell columns, dividing Hofbauer cells in villus cores, and invasive cytotrophoblasts, but frequencies differed. Cytotrophoblasts in explants infected by Nicaraguan strains were invasive, whereas those infected by prototype MR766 largely remained in cell columns, and titers varied by donor and strain. In basal decidua, ZIKV replicated in glandular epithelium, decidual cells, and immune cells. ZIKV-infected AmEpCs frequently occurred in pairs and expressed Ki67 and phosphohistone H3, indicating replication in dividing cells. CONCLUSIONS: ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.

Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

The emergence of congenital Zika virus (ZIKV) disease, with its devastating effects on the fetus, has prompted development of vaccines and examination of how ZIKV breaches the maternal-fetal barrier. Infection of placental and decidual tissue explants has demonstrated cell types at the uterine-placental interface susceptible to infection and suggests routes for transmission across the placenta and amniochorionic membrane. ZIKV replicates in proliferating Hofbauer cells within chorionic villi in placentas from severe congenital infection. Explants of anchoring villi recapitulate placental architecture and early-stage development and suggest infected Hofbauer cells disseminate virus to fetal blood vessels. ZIKV infection of explants represents a surrogate human model for evaluating protection against transmission by antibodies in vaccine recipients and passive immune formulations and novel therapeutics.

Human cytomegalovirus (HCMV) is the major viral cause of congenital infection and birth defects. Primary maternal infection often results in virus transmission, and symptomatic babies can have permanent neurological deficiencies and deafness. Congenital infection can also lead to intrauterine growth restriction, a defect in placental transport. HCMV replicates in primary cytotrophoblasts (CTBs), the specialized cells of the placenta, and inhibits differentiation/invasion. Human trophoblast progenitor cells (TBPCs) give rise to the mature cell types of the chorionic villi, CTBs and multi-nucleated syncytiotrophoblasts (STBs). Here we report that TBPCs are fully permissive for pathogenic and attenuated HCMV strains. Studies with a mutant virus lacking a functional pentamer complex (gH/gL/pUL128-131A) showed that virion entry into TBPCs is independent of the pentamer. In addition, infection is blocked by a potent human neutralizing monoclonal antibody (mAb), TRL345, reactive with glycoprotein B (gB), but not mAbs to the pentamer proteins pUL130/pUL131A. Functional studies revealed that neutralization of infection preserved the capacity of TBPCs to differentiate and assemble into trophospheres composed of CTBs and STBs in vitro. Our results indicate that mAbs to gB protect trophoblast progenitors of the placenta and could be included in antibody treatments developed to suppress congenital infection and prevent disease.

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, including microcephaly, neurological deficits, hearing impairment, and vision loss. We previously reported that epithelial cells in amniotic membranes of placentas from newborns with intrauterine growth restriction and underlying congenital HCMV infection contain viral proteins in cytoplasmic vesicles. Herein, we immunostained amniotic membranes from 51 placentas from symptomatic and asymptomatic congenital infection with HCMV DNA in amniotic fluid and/or newborn saliva, intrauterine growth restriction, preterm deliveries, and controls. We consistently observed HCMV proteins in amniotic epithelial cells (AmEpCs) from infected placentas, sometimes with aberrant morphology. Primary AmEpCs isolated from mid-gestation placentas infected with pathogenic VR1814 proliferated and released infectious progeny for weeks, producing higher virus titers than late-gestation cells that varied by donor. In contrast to intact virion assembly compartments in differentiated retinal pigment epithelial cells, infected AmEpCs made dispersed multivesicular bodies. Primary AmEpCs and explants of amniochorionic membranes from mid-gestation placentas formed foci of infection, and interferon-β production was prolonged. Infected AmEpCs up-regulated anti-apoptotic proteins survivin and Bcl-x L by mechanisms dependent and independent of the activated STAT3. Amniotic membranes naturally expressed both survivin and Bcl-x L , indicating that fetal membranes could foster persistent viral infection. Our results suggest strengthening innate immune responses and reducing viral functions could suppress HCMV infection in the fetal compartment.

Human cytomegalovirus (HCMV) infection is transmitted from the infected mother to the placenta and fetus. Virus replicates in the decidua, invasive cytotrophoblasts that breach the uterine vasculature and villous cytotrophoblasts underlying syncytiotrophoblasts, then reaches blood vessels in the villus core. Virus replication, fibrosis, and edema result in a hypoxic intrauterine environment and release of cytokines that stimulates compensatory development of the placenta. We employed villous explant cultures to study viral effects on differentiation and test novel approaches to rescue the placenta from infection.

We investigated human cytomegalovirus pathogenesis by comparing infection with the low-passage, endotheliotropic strain VR1814 and the attenuated laboratory strain AD169 in human placental villi as explants in vitro and xenografts transplanted into kidney capsules of SCID mice (ie, mice with severe combined immunodeficiency). In this in vivo human placentation model, human cytotrophoblasts invade the renal parenchyma, remodel resident arteries, and induce a robust lymphangiogenic response. VR1814 replicated in villous and cell column cytotrophoblasts and reduced formation of anchoring villi in vitro. In xenografts, infected cytotrophoblasts had a severely diminished capacity to invade and remodel resident arteries. Infiltrating lymphatic endothelial cells proliferated, aggregated, and failed to form lymphatic vessels. In contrast, AD169 grew poorly in cytotrophoblasts in explants, and anchoring villi formed normally in vitro. Likewise, viral replication was impaired in xenografts, and cytotrophoblasts retained invasive capacity, but some partially remodeled blood vessels incorporated lymphatic endothelial cells and were permeable to blood. The expression of both vascular endothelial growth factor (VEGF)-C and basic fibroblast growth factor increased in VR1814-infected explants, whereas VEGF-A and soluble VEGF receptor-3 increased in those infected with AD169. Our results suggest that viral replication and paracrine factors could undermine vascular remodeling and cytotrophoblast-induced lymphangiogenesis, contributing to bleeding, hypoxia, and edema in pregnancies complicated by congenital human cytomegalovirus infection.

Summary Zika virus (ZIKV) infection during pregnancy is linked to severe birth defects, but mother-to-fetus transmission routes are unknown. We infected different primary cell types from mid- and late-gestation placentas and explants from first-trimester chorionic villi with the prototype Ugandan and a recently isolated Nicaraguan ZIKV strain. ZIKV infects primary human placental cells and explants—cytotrophoblasts, endothelial cells, fibroblasts, and Hofbauer cells in chorionic villi and amniotic epithelial cells and trophoblast progenitors in amniochorionic membranes—that express Axl, Tyro3, and/or TIM1 viral entry cofactors. ZIKV produced NS3 and E proteins and generated higher viral titers in amniotic epithelial cells from mid-gestation compared to late-gestation placentas. Duramycin, a peptide that binds phosphatidylethanolamine in enveloped virions and precludes TIM1 binding, reduced ZIKV infection in placental cells and explants. Our results suggest that ZIKV spreads from basal and parietal decidua to chorionic villi and amniochorionic membranes and that targeting TIM1 could suppress infection at the uterine-placental interface.

Human cytomegalovirus (HCMV) is the major viral cause of birth defects worldwide. Affected infants can have temporary symptoms that resolve soon after birth, such as growth restriction, and permanent disabilities, including neurological impairment. Passive immunization of pregnant women with primary HCMV infection is a promising treatment to prevent congenital disease. To understand the effects of sustained viral replication on the placenta and passive transfer of protective antibodies, we performed immunohistological analysis of placental specimens from women with untreated congenital infection, HCMV-specific hyperimmune globulin treatment, and uninfected controls. In untreated infection, viral replication proteins were found in trophoblasts and endothelial cells of chorionic villi and uterine arteries. Associated damage included extensive fibrinoid deposits, fibrosis, avascular villi, and edema, which could impair placental functions. Vascular endothelial growth factor and its receptor fms-like tyrosine kinase 1 (Flt1) were up-regulated, and amniotic fluid contained elevated levels of soluble Flt1 (sFlt1), an antiangiogenic protein, relative to placental growth factor. With hyperimmune globulin treatment, placentas appeared uninfected, vascular endothelial growth factor and Flt1 expression was reduced, and sFlt1 levels in amniotic fluid were lower. An increase in the number of chorionic villi and blood vessels over that in controls suggested compensatory development for a hypoxia-like condition. Taken together the results indicate that antibody treatment can suppress HCMV replication and prevent placental dysfunction, thus improving fetal outcome.

How human cytomegalovirus (CMV) reaches the fetus across the placenta is unknown. The major viral cause of congenital disease, CMV infects the uterine-placental interface with varied outcomes depending on the strength of maternal humoral immunity and gestational age. Covering the surface of villi that float in blood, syncytiotrophoblasts express the neonatal Fc receptor (FcRn) that transports IgG for passive immunity. Immunohistochemical analysis of early-gestation biopsy specimens showed an unusual pattern of CMV replication proteins in underlying villus cytotrophoblasts, whereas syncytiotrophoblasts were spared. Found in placentas with low to moderate CMV-neutralizing antibody titers, this pattern suggested virion transcytosis across the surface. In contrast, syncytiotrophoblasts from placentas with high neutralizing titers contained viral DNA and caveolin-1-positive vesicles in which IgG and CMV glycoprotein B co-localized. In villus explants, IgG-virion transcytosis and macrophage uptake were blocked with trypsin-treatment and soluble protein A. Quantitative analysis in polarized epithelial cells showed that FcRn-mediated transcytosis was blocked by the Fc fragment of IgG, but not F(ab')(2). Our results suggest that CMV virions could disseminate to the placenta by co-opting the receptor-mediated transport pathway for IgG. These findings could explain the efficacy of hyperimmune IgG for treatment of primary CMV infection during gestation and support vaccination.

During human gestation, viruses can cause intrauterine infections associated with pregnancy complications and fetal abnormalities. The ability of viruses to spread from the infected mother to the fetus arises from the architecture of the placenta, which anchors the fetus to the uterus. Placental cytotrophoblasts differentiate, assume an endothelial phenotype, breach uterine blood vessels and form a hybrid vasculature that amplifies the maternal blood supply for fetal development. Human cytomegalovirus - the major cause of congenital disease - infects the uterine wall and the adjacent placenta, suggesting adaptation for pathogen survival in this microenvironment. Infection of villus explants and differentiating and/or invading cytotrophoblasts offers an in vitro model for studying viruses associated with prenatal infections.

Background. Human cytomegalovirus (HCMV) is the major viral etiology of congenital infection and birth defects. Fetal transmission is high (30%–40%) in primary maternal infection, and symptomatic babies have permanent neurological, hearing, and vision defects. Recurrent infection is infrequently transmitted (2%) and largely asymptomatic. Congenital infection is also associated with intrauterine growth restriction (IUGR). Methods. To investigate possible underlying HCMV infection in cases of idiopathic IUGR, we studied maternal and cord sera and placentas from 19 pregnancies. Anti-HCMV antibodies, hypoxia-related factors, and cmvIL-10 were measured in sera. Placental biopsy specimens were examined for viral DNA, expression of infected cell proteins, and pathology. Results. Among 7 IUGR cases, we identified 2 primary and 3 recurrent HCMV infections. Virus replicated in glandular epithelium and lymphatic endothelium in the decidua, cytotrophoblasts, and smooth muscle cells in blood vessels of floating villi and the chorion. Large fibrinoids with avascular villi, edema, and inflammation were significantly increased. Detection of viral proteins in the amniotic epithelium indicated transmission in 2 cases of IUGR with primary infection and 3 asymptomatic recurrent infections. Conclusions. Congenital HCMV infection impairs placental development and functions and should be considered as an underlying cause of IUGR, regardless of virus transmission to the fetus.

Cytomegalovirus (CMV) is the major cause of congenital infection and disease leading to permanent birth defects. In about 35-40% of pregnant women with primary CMV infection, virus crosses the placenta, resulting in the birth of congenitally infected babies. In contrast, this happens in only 1-3% of seropositive women with strong CMV-specific humoral immunity. Whether CMV reaches the fetus and disseminates depends on the level of high-avidity antibodies in the maternal circulation and the passive immunity of the fetus.To identify CMV infection in uncomplicated deliveries based on detection of viral DNA in placental biopsy specimens at term. To quantify CMV-specific IgG avidity, neutralizing titer, IgG1 concentration, and characterize the immunoblot profiles for CMV proteins in paired samples of placental and cord blood sera.In accord with earlier reports, CMV DNA was detected in 39% (11/28) of placentas with mean- to high-avidity CMV-specific IgG. In seropositive women, the concentration of antiviral antibodies, specifically IgG1, increased in the fetal bloodstream, and CMV neutralizing titers in maternal and fetal blood were comparable.CMV-specific, high-avidity neutralizing antibodies from maternal circulation are transcytosed to the fetal bloodstream, contribute to suppression of viral replication in the placenta and could prevent congenital disease.

Human cytomegalovirus (CMV) infection is a major cause of morbidity in immunosuppressed individuals, and congenital CMV infection is a leading cause of birth defects in newborns. Infection with pathogenic viral strains alters cell-cell and cell-matrix interactions, affecting extracellular matrix remodeling and endothelial cell migration. The multifunctional cytokine transforming growth factor (TGF)-beta1 regulates cell proliferation, differentiation, and extracellular matrix remodeling. Secreted as a latent protein complex, TGF-beta1 requires activation before binding to receptors that phosphorylate intracellular effectors. TGF-beta1 is activated by integrin alphavbeta6, which is strongly induced in the epithelium by injury and inflammation but has not previously been found in endothelial cells. Here, we report that CMV infection induces integrin alphavbeta6 expression in endothelial cells, leading to activation of TGF-beta1, signaling through its receptor ALK5, and phosphorylation of its intracellular effector Smad3. Infection of endothelial cells was also found to stimulate collagen synthesis through a mechanism dependent on both TGF-beta1 and integrin alphavbeta6. Immunohistochemical analysis showed integrin alphavbeta6 up-regulation in capillaries proximal to foci of CMV infection in lungs, salivary glands, uterine decidua, and injured chorionic villi of the placenta, demonstrating both its induction in endothelium and up-regulation in epithelium in vivo. Our results suggest that activation of TGF-beta1 by integrin alphavbeta6 contributes to pathological changes and may impair endothelial cell functions in tissues that are chronically infected with CMV.

Human cytomegalovirus (HCMV) is the most common cause of congenital viral infection. Affected children can have permanent neurological complications, including hearing loss, visual impairment and mental retardation1–3. In Australia, 57% of women are seronegative and at risk for primary infection and transmission of virus to the fetus during pregnancy4. Despite its public health significance, the specific molecular and cellular basis of HCMV replication in the human placenta and pathogenesis associated with poor clinical outcome are unknown. Direct fetal infection is involved in severe cases of neuropathology and infection of the placenta can impair its development and functions resulting in a hypoxic environment5–8 and stillbirth6,9,10. Gestational age at the time of infection is an important determinant of outcome. The rates of virus transmission increase from 30% in first trimester to over 70% in third trimester suggesting different mechanisms for overcoming the placental barrier2. Remarkable insights into viral pathogenesis factors that function in the tissue environment have been gained by studying congenitally infected placentas and explants infected by clinical strains ex vivo. Together these studies revealed that direct infection of specialised placental cells and paracrine factors contribute to impaired development and functional defects.

Studies of intrauterine human cytomegalovirus (CMV) infection have shown suppressed replication in the decidua and placenta of strongly seropositive women. Biopsy specimens often contain CMV virion glycoprotein B and DNA in syncytiotrophoblasts and villus core macrophages without productive infection. Focal replication occurs in placentas of women with low to moderate neutralizing antibody titres. Infected cytotrophoblasts downregulate key adhesion and immune molecules required for invasiveness and maternal immune tolerance and reduce matrix metalloproteinase-9 protein and activity, impairing degradation of the extracellular matrix. Here, we used flow cytometry and quantitative RT-PCR analyses to quantify differentiation molecules expressed in freshly isolated cytotrophoblasts purified from placentas at term and differentiating cells infected in vitro with VR1814, a pathogenic clinical strain. Cell surface proteins including E-cadherin, VE-cadherin, HLA-G, and CMV receptors--epidermal growth factor receptor and integrins beta1 and alphavbeta3--were expressed on purified cells, as were integrins alpha9 and beta6, which were not previously studied. Infected cytotrophoblasts dysregulate the levels of particular cell-matrix and cell-cell adhesion proteins and their transcripts. CMV replication in late gestation placentas with considerable reserves could deplete cytotrophoblast progenitors, thereby impairing syncytiotrophoblast development and increasing the risk of virus transmission to fetal blood vessels.

Aneuploid cells in the placenta are associated with poor pregnancy outcomes, but the mechanisms are unclear. Here, we examined the cytotrophoblast (CTB) differentiation pathway that leads to uterine invasion in pregnancies complicated by trisomy 21 (T21) as compared with their normal counterparts. Surprisingly, we observed a wide spectrum of T21 effects. Morphologically, some samples appeared near normal, while others had extensive fibrinoid deposition and apoptosis of CTBs at the maternal–fetal interface (confirmed by TUNEL labeling). At a molecular level, the cells' expression of stage-specific molecules was variably misregulated. At one end of the spectrum, samples with less apoptosis had relatively normal staining patterns. At the other end, samples with extensive apoptosis showed significantly decreased staining for these antigens. Additional studies confirmed that the effects we observed had functional consequences, because the cells exhibited marked phenotypic alterations in vitro, including a large increase in MMP-9 production, which distinguishes the effects of T21 on CTBs from those of preeclampsia. The morphologic, phenotypic, and functional differences among CTBs from pregnancies complicated by T21 illustrate the importance of the interplay between fetal/placental genotype and maternal influences on pregnancy outcome. Furthermore, our data may explain why a significant number of these pregnancies end in spontaneous abortion while others survive to term. © 2004 Wiley-Liss, Inc.

A novel and rapid EBNA-1 titration method has been developed which uses immunoprecipitation of specific DNA-protein complexes with EBNA-1-positive serum. The method is more sensitive than the conventional immunofluorescence method and has potential value as a diagnostic reagent for clinical laboratories. TPA induction of putative anti-EBNA-1 protein of cellular origin is discussed, which may play a key role for the shift from latent to lytic replication of EBV.

Four transcripts belonging to the EBNA-3 family were detected during immortalization of human tonsillar lymphocytes with the Epstein-Barr virus. These four transcripts were also detected in immortalized B-lymphoblastoid cells (LCL) and the Burkitt's lymphoma cell line, Namalwa. A 5.0-kb transcript hybridized with the EBNA-3A probe; a 8.5-kb transcript hybridized with the EBNA-3B and -3C probes; and a 5.6- and a 3.6-kb transcript hybridized with the EBNA-3C probe. Sequencing and cDNA analysis showed that the EBNA-3C transcripts contained exons of the most rightward Bam Hl-E open reading frames, BERF-3 and BERF-4. Moreover, sequencing of cDNA clones revealed exons in the 5′ portion homologous to regions in EBV Bam Hl-C, -W, and -U. Hybridization experiments with EBV Bam HI-W and -U probes identified the 8.5- and 5.6-kb transcripts but not the 3.6-kb transcript in LCL. Organization of these transcripts from the EBNA-3 family is discussed.

The transcription of latent Epstein-Barr viral (EBV) genes, i.e., genes encoding EBNA-1, EBNA-2, EBNA-3A, -3B, -3C, and LMP, were detected in human tonsillar lymphocytes early after infection with EBV. Transcription of the BHLF-1 open reading frame was also detected at this initial phase of immortalization. Cycloheximide treatment inhibited the transcription of all the latent EBV genes but not BHLF-1. These results suggest that BHLF-1 might be considered an immediate-early gene of EBV. Cycloheximide treatment of EBV-infected cells reduced not only the degree of the transcription but also the size of the transcript for EBNA-2. We hypothesize that the immediate-early expression of BHLF-1 may be required for the enhanced transcription of the viral genes in lymphocytes early after infection with EBV.