Human cytomegalovirus (CMV) dysregulates neurodevelopmental pathways in cerebral organoids.

Human cytomegalovirus (CMV) infection is the leading non-genetic aetiology of congenital malformation in developed countries, causing significant fetal neurological injury. This study investigated potential CMV pathogenetic mechanisms of fetal neural malformation using in vitro human cerebral organoids. Cerebral organoids were permissive to CMV replication, and infection dysregulated cellular pluripotency and differentiation pathways. Aberrant expression of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK), sonic hedgehog (SHH), pluripotency, neurodegeneration, axon guidance, hippo signalling and dopaminergic synapse pathways were observed in CMV-infected organoids using immunofluorescence and RNA-sequencing. Infection with CMV resulted in dysregulation of 236 Autism Spectrum Disorder (ASD)-related genes (p = 1.57E-05) and pathways. This notable observation suggests potential links between congenital CMV infection and ASD. Using DisGeNET databases, 103 diseases related to neural malformation or mental disorders were enriched in CMV-infected organoids. Cytomegalovirus infection-related dysregulation of key cerebral cellular pathways potentially provides important, modifiable pathogenetic mechanisms for congenital CMV-induced neural malformation and ASD.

Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.

Establishing the relationship between non-human primates and mangrove forests at the global, national, and local scales.

Global and spatially explicit information about the interaction between habitat and wildlife species is critical to enhancing conservation efforts. Despite the recognized importance of mangrove forests to non-human primates, the relationship between the two lacks understanding. To counter this, we created the MangPrim-21 database to map and measure the locations of interactions between all non-human primates and all mangrove forests globally. We report our findings across the global, national, and local scales for all inventoried non-human primates and all inventoried mangrove forests. Globally, we find that half of all non-primates potentially use mangrove forests, and more than half of the global mangrove forest falls within the delineated range of at least one non-human primate species. Nationally, we find that Indonesia, Madagascar, Brazil, Cameroon, and Malaysia likely have the most non-human primate and mangrove forest interactions. At the subnational level, we find that several discrete locations in Kalimantan are critical to both mangrove forests and non-human primates. The MangPrim-21 database provides a globally consistent and locally applicable database of non-human primate and mangrove forest interactions. The results presented have broader implications for non-human primate and mangrove conservation and global actions to protect both. Additionally, our results raise questions about the idea that non-human primates primarily use mangrove forests as a refuge from human encroachment and habitat degradation.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H.

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.

High-resolution bathymetries and shorelines for the Great Lakes of the White Nile basin.

HRBS-GLWNB 2020 presents the first open-source and high-resolution bathymetry, shoreline, and water level data for Lakes Victoria, Albert, Edward, and George in East Africa. For each Lake, these data have three primary products collected for this project. The bathymetric datasets were created from approximately 18 million acoustic soundings. Over 8,200 km of shorelines are delineated across the three lakes from high-resolution satellite systems and uncrewed aerial vehicles. Finally, these data are tied together by creating lake surface elevation models collected from GPS and altimeter measures. The data repository includes additional derived products, including surface areas, water volumes, shoreline lengths, lake elevation levels, and geodetic information. These data can be used to make allocation decisions regarding the freshwater resources within Africa, manage food resources on which many tens of millions of people rely, and help preserve the region's endemic biodiversity. Finally, as these data are tied to globally consistent geodetic models, they can be used in future global and regional climate change models.

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity.

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.

Mangroves and coastal topography create economic "safe havens" from tropical storms.

Evidence suggests that mangroves protect economic activity in coastal areas. We estimate this protection from mangroves and coastal elevation globally, examining both "direct" and "indirect" exposure events (< 100 km vs. ≥ 100 km distance from a cyclone's "eye", respectively). We find that higher elevation (≥ 50 m) or wide mangroves (≥ 10 m seaward width) alone shelter economic activity from indirect cyclone exposure, whereas protection from direct cyclone exposure occurs only in high elevation communities with wide mangroves. Our results reveal that the majority of these "safe havens" are in upper middle-income countries but provide significant benefits to populations in lower middle-income countries.

A placental specific miRNA miR-517a-3p exerts anti-human cytomegalovirus activity.

Human cytomegalovirus congenital infection is the leading non-genetic cause of fetal malformation in developed countries. There are currently no safe antivirals for use during pregnancy. Placental trophoblast cells specifically secrete exosomes containing miRNA from the chromosome 19 miRNA cluster (C19MC) which confer viral resistance to recipient cells. We show the highly expressed C19MC miRNA miR-517a-3p inhibits HCMV replication and viral protein expression in both fibroblast and trophoblast cell cultures (71.6% and 50.4% inhibition of HCMV DNA at 7 days post infection respectively; p < 0.05). This naturally occurring molecule has potential for opening-up antiviral therapeutic strategies for pregnancy.

Receptors in host pathogen interactions between human cytomegalovirus and the placenta during congenital infection.

Cellular receptors in human cytomegalovirus (HCMV) mother to child transmission play an important role in congenital infection. Placental trophoblast cells are a significant cell type in placental development, placental functional processes, and in HCMV transmission. Different cells within the placental floating and chorionic villi present alternate receptors for HCMV cell entry. Syncytiotrophoblasts present neonatal Fc receptors that bind and transport circulating maternal immunoglobulin G across the placental interface which can also be bound to HCMV virions, facilitating viral entry into the placenta and foetal circulation. Cytotrophoblast express HCMV receptors including integrin-α1ß1, integrin-αVß3, epidermal growth factor receptor and platelet-derived growth factor receptor alpha. The latter interacts with HCMV glycoprotein-H, glycoprotein-L and glycoprotein-O (gH/gL/gO) trimers (predominantly in placental fibroblasts) and the gH/gL/pUL128, UL130-UL131A pentameric complex in other placental cell types. The pentameric complex allows viral tropism of placental trophoblasts, endothelial cells, epithelial cells, leukocytes and monocytes. This review outlines HCMV ligands and target receptor proteins in congenital HCMV infection.

Investigational Antiviral Therapy Models for the Prevention and Treatment of Congenital Cytomegalovirus Infection during Pregnancy.

Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation, lifelong disease, and, in severe cases, fetal or neonatal death. Placental infection with HCMV is the major mechanism of mother-to-child transmission (MTCT) and fetal injury. Thus, any pharmaceutical antiviral interference to reduce viral load may reduce placental damage, MTCT, and fetal disease. However, there is currently no licensed HCMV antiviral for use during pregnancy. In this study, aciclovir and the HCMV-specific antivirals letermovir, maribavir, and cidofovir were compared with ganciclovir for antiviral effects in model systems of pregnancy, including first-trimester TEV-1 trophoblast cell cultures and third-trimester ex vivo placental explant histocultures. HCMV-infected trophoblasts at 7 days postinfection (dpi) showed an EC50 of 21 µM for aciclovir, 0.0007 µM for letermovir, 0.11 µM for maribavir, and 0.29 µM for cidofovir, relative to 0.42 µM for ganciclovir. Antivirals added at 10 µM showed no cytotoxic effects and did not affect trophoblast cell proliferation (P > 0.9999). Multiple-round HCMV replication measured at 7 dpi showed letermovir, maribavir, and cidofovir treatment inhibited immediate early, early, and true late viral protein expression as assayed on Western blots. Antiviral treatment of HCMV-infected placental explants showed significant inhibition (P < 0.05) of viral replication with letermovir (83.3%), maribavir (83.6%), cidofovir (89.3%), and ganciclovir (82.4%), but not aciclovir (P > 0.9999). In ex vivo model systems, recently trialed HCMV antivirals letermovir and maribavir were effective at inhibiting HCMV replication. They partly fulfil requirements for use as safe and effective therapeutics during pregnancy to control congenital HCMV. Clinical trials of these newer agents would assist assessment of their utility in pregnancy.

A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus.

Nuclear egress is a rate-limiting step of herpesviral replication, restricting the nucleocytoplasmic transport of viral capsids. The process is regulated by two viral nuclear egress proteins (core NEC pUL50-pUL53), which recruit additional cellular and viral proteins. The multicomponent NEC mediates disassembly of the nuclear lamina barrier and the docking of nuclear capsids. The quantitation of nuclear egress has been accomplished by electron microscopic analysis, but is generally hampered by the low number of detectable cytoplasmic capsids. A newly established method for the quantitation of viral nuclear egress improves the characterization of viral mutants, host cell permissiveness and antiviral drug efficacy. In this study, various strains of human cytomegalovirus (HCMV) were used to measure the replication efficiencies in primary human fibroblasts, applying methods of cell fractionation, DNase digestion, sucrose cushions and quantitative PCR. Several stages of optimization led to a reliable quantitative assay that allowed the characterization of viral nuclear egress efficacy. Using this assay, recovery of the nuclear egress of a NEC-defective HCMV mutant was quantitatively assessed by applying an inducible NEC-expressing fibroblast culture for trans-complementation. This novel assay system can be further used to accurately quantitate and characterize the functionality of nuclear egress of HCMV or other herpesviruses.

Tenth Scientific Biennial Meeting of the Australasian Virology Society-AVS10 2019.

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2-5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on "Successfully transitioning from post-doc to lab head", winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.

Fetal therapies for cytomegalovirus: What we tell prospective parents.

Congenital CMV is the most common congenital infection in the developed world. Infection results in congenital disease ranging from asymptomatic infection to severe neurodevelopmental impairment, and occasionally fetal or neonatal death. Fetal infection can occur through maternal-fetal transmission during primary maternal infection or maternal reactivation or re-infection. Awareness among maternal health care providers and parents is low. The prevention of maternal CMV infection currently relies on hygiene measures, with no effective CMV vaccine or prophylactic therapies. No licensed treatment options are available to prevent maternal-fetal transmission or fetal disease. Hyperimmunoglobulin and valaciclovir have been investigated for prevention of maternal-fetal transmission or fetal treatment, with some evidence supporting consideration of maternal administration of hyperimmunoglobulin or valaciclovir therapy in certain circumstances. This article outlines the clinical evidence regarding proven preventative behavioral measures and experimental hyperimmunoglobulin and valaciclovir therapies, that is structured around common questions asked by pregnant women about CMV infection. It is aimed to help maternity health care providers counsel prospective parents about congenital CMV disease and the preventative and therapeutic strategies currently available.

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.

Patient-Derived Cytomegaloviruses with Different Ganciclovir Sensitivities from UL97 Mutation Retain Their Replication Efficiency and Some Kinase Activity In Vitro.

Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro.

Mangroves shelter coastal economic activity from cyclones.

Mangroves shelter coastlines during hazardous storm events with coastal communities experiencing mangrove deforestation are increasingly vulnerable to economic damages resulting from cyclones. To date, the benefits of mangroves in terms of protecting coastal areas have been estimated only through individual case studies of specific regions or countries. Using spatially referenced data and statistical methods, we track from 2000 to 2012 the impact of cyclones on economic activity in coastal regions inhabited by nearly 2,000 tropical and subtropical communities across 23 major mangrove-holding countries. We use nighttime luminosity to represent temporal trends in coastal economic activity and find that direct cyclone exposure typically results in permanent loss of 5.4-6.7 mo for a community with an average mangrove extent (6.3 m per meter of coastline); whereas, a community with more extensive mangroves (25.6 m per meter of coastline) experiences a loss equivalent to 2.6-5.5 mo. These results suggest that mangrove restoration efforts for protective benefits may be more cost effective, and mangrove deforestation more damaging, than previously thought.

The use of open source GIS algorithms, big geographic data, and cluster computing techniques to compile a geospatial database that can be used to evaluate upstream bathing and sanitation behaviours on downstream health outcomes in Indonesia, 2000-2008.

BACKGROUND: Waterborne diseases are one of the leading causes of mortality in developing countries, and diarrhea alone is responsible for over 1.5 million deaths annually. Such waterborne illnesses most often affect those in impoverished rural communities who rely on rivers for their supply of drinking water. Deaths are most common among infants and the elderly. Without knowledge of which communities are upstream of a community, upstream sanitary and bathing behaviors can never be directly linked to downstream health outcomes including disease outbreaks. Although current GIS technologies can answer the upstream question for a limited number of downstream communities, no systematic way existed of labeling each downstream village with all its upstream contributing villages along river networks or within basins at the large national scale, such as in Indonesia. This limitation prohibits macro analyses of waterborne illness across developing world communities globally. RESULTS: This novel method approach combines parallel computing, big data, community data, and open source GIS to create a database of upstream communities for 50,000-70,0000 villages in Indonesia across four differing periods. The resultant village database provides information that can be tied to the Indonesian PODES health and behavior surveys in each village to connect upstream sanitary behaviors to downstream health outcomes. We find that the approximately 250,000 communities analyzed across the four periods in Indonesia have a combined total of 13.7 million upstream villages. The average number of upstream villages per village was almost 55, the maximum number of upstream villages for any single village was over 5300. CONCLUSIONS: Advances in big-data availability, particularly high-resolution elevation data, the lowering of the cost of parallel computing options, mass survey data, and open source GIS algorithms that can utilize parallel processing and big-data, open new opportunities for the study of human health at micro granularities but across entire nations. The database generated has already been used by health researchers to compute the influence of upstream behaviors on downstream diarrhea outbreaks and to monitor avoidance behaviors to upstream water behaviors across all downstream 250,000 Indonesian villages over 4 years, and further waterborne health analyses are underway.