Initiation of Metformin in Early Pregnancy Results in Fetal Bioaccumulation, Growth Restriction & Renal Dysmorphology in a Primate Model.

BACKGROUND: In recent years, pragmatic metformin use in pregnancy has stretched to include prediabetes, type 2 diabetes, gestational diabetes and (most recently) pre-eclampsia. With its expanded use, however, concerns of unintended harm have been raised. OBJECTIVE: We developed an experimental primate model and applied triple-quadruple pole LC mass spectrometry (UHPLC-QQQ) for direct quantitation of maternal and fetal tissue metformin levels with detailed fetal biometry and histopathology. STUDY DESIGN: Within 30 days of confirmed conception (defined as early pregnancy), n=13 time-bred (TMB) Rhesus dams with gestations designated for fetal necropsy were initiated on twice daily human dose-equivalent 10 mg/kg metformin or vehicle control. Pregnant dams were maintained as pairs and fed either a control chow or 36% fat Western-style diet (WSD). Metformin or placebo vehicle control were delivered in a variety of treats while animals were separated via a slide. A Cesarean was performed at G145, and amniotic fluid and blood were collected and the fetus and placenta were delivered. The fetus was immediately necropsied by trained primate center personnel. All fetal organs were dissected, measured, sectioned, and processed per clinical standards. Fluid and tissue metformin levels were assayed using validated UHPLC-QQQ in SRM against standard curves. RESULTS: Among the n=13 G145 pregnancies with fetal necropsy, n=1 dam and its fetal tissues had detectable metformin levels despite being allocated to the vehicle control group (>1 µM metformin/kg maternal weight or fetal/placental tissue), while a second fetus allocated to the vehicle control group had severe fetal growth restriction (birthweight 248.32 g, <1%) and was suspected of having a fetal congenital condition. After excluding these two fetal gestations from further analyses, 11 fetuses from dams initiated on either vehicle control (n=4, 3 female, 1 male fetuses) or 10 mg/kg metformin (n=7, 5 female, 2 male fetuses) were available for analyses. Among dams initiated on metformin by G30 (regardless of maternal diet), we observed significant bioaccumulation within the fetal kidney (0.78-6.06 µmol/kg, mean 2.48 µmol/kg) , liver (0.16-0.73 µmol/kg, mean 0.38 µmol/kg), fetal gut (0.28-1.22 µmol/kg, mean 0.70 µmol/kg), amniotic fluid (0.43-3.33 µmol/L, mean 1.88 µmol/L), placenta (0.16-1.0 µmol/kg , mean 0.50 µmol/kg) and fetal serum (0 -0.66 µmol/L , mean 0.23 µmol/L ), and fetal urine (4.1-174.1 µmol/L mean 38.5 µmol/L ), with fetal levels near biomolar equivalent to maternal levels (maternal serum 0.18-0.86 µmol/L , mean 0.46 µmol/L; maternal urine 42.6-254.0 µmol/L , mean 149.3 µmol/L). WSD feeding neither accelerated nor reduced metformin bioaccumulations in maternal or fetal serum, urine, amniotic fluid, placenta nor fetal tissues. In these 11 animals, fetal bioaccumulation of metformin was associated with less fetal skeletal muscle (57% lower cross-sectional area of gastrocnemius) and decreased liver, heart, and retroperitoneal fat masses (p<0.05), collectively driving lower delivery weight (p<0.0001) without changing the crown-rump length. Sagittal sections of fetal kidneys demonstrated delayed maturation, with disorganized glomerular generations and increased cortical thickness; this renal dysmorphology was not accompanied by structural nor functional changes indicative of renal insufficiency. CONCLUSIONS: We demonstrate fetal bioaccumulation of metformin with associated fetal growth restriction and renal dysmorphology following maternal initiation of the drug within 30 days of conception in primates. Given these results and the prevalence of metformin use during pregnancy, additional investigation of any potential immediate and enduring effects of prenatal metformin use is warranted.

Discrete placental gene expression signatures accompany diabetic disease classifications during pregnancy.

BACKGROUND: Gestational diabetes mellitus affects up to 10% of pregnancies and is classified into subtypes gestational diabetes subtype A1 (GDMA1) (managed by lifestyle modifications) and gestational diabetes subtype A2 (GDMA2) (requiring medication). However, whether these subtypes are distinct clinical entities or more reflective of an extended spectrum of normal pregnancy endocrine physiology remains unclear. OBJECTIVE: Integrated bulk RNA-sequencing (RNA-seq), single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomics harbors the potential to reveal disease gene signatures in subsets of cells and tissue microenvironments. We aimed to combine these high-resolution technologies with rigorous classification of diabetes subtypes in pregnancy. We hypothesized that differences between preexisting type 2 and gestational diabetes subtypes would be associated with altered gene expression profiles in specific placental cell populations. STUDY DESIGN: In a large case-cohort design, we compared validated cases of GDMA1, GDMA2, and type 2 diabetes mellitus (T2DM) to healthy controls by bulk RNA-seq (n=54). Quantitative analyses with reverse transcription and quantitative PCR of presumptive genes of significant interest were undertaken in an independent and nonoverlapping validation cohort of similarly well-characterized cases and controls (n=122). Additional integrated analyses of term placental single-cell, single-nuclei, and spatial transcriptomics data enabled us to determine the cellular subpopulations and niches that aligned with the GDMA1, GDMA2, and T2DM gene expression signatures at higher resolution and with greater confidence. RESULTS: Dimensional reduction of the bulk RNA-seq data revealed that the most common source of placental gene expression variation was the diabetic disease subtype. Relative to controls, we found 2052 unique and significantly differentially expressed genes (-22 thresholds; q<0.05 Wald Test) among GDMA1 placental specimens, 267 among GDMA2, and 1520 among T2DM. Several candidate marker genes (chorionic somatomammotropin hormone 1 [CSH1], period circadian regulator 1 [PER1], phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta [PIK3CB], forkhead box O1 [FOXO1], epidermal growth factor receptor [EGFR], interleukin 2 receptor subunit beta [IL2RB], superoxide dismutase 3 [SOD3], dedicator of cytokinesis 5 [DOCK5], suppressor of glucose, and autophagy associated 1 [SOGA1]) were validated in an independent and nonoverlapping validation cohort (q<0.05 Tukey). Functional enrichment revealed the pathways and genes most impacted for each diabetes subtype, and the degree of proximal similarity to other subclassifications. Surprisingly, GDMA1 and T2DM placental signatures were more alike by virtue of increased expression of chromatin remodeling and epigenetic regulation genes, while albumin was the top marker for GDMA2 with increased expression of placental genes in the wound healing pathway. Assessment of these gene signatures in single-cell, single-nuclei, and spatial transcriptomics data revealed high specificity and variability by placental cell and microarchitecture types. For example, at the cellular and spatial (eg, microarchitectural) levels, distinguishing features were observed in extravillous trophoblasts (GDMA1) and macrophages (GDMA2). Lastly, we utilized these data to train and evaluate 4 machine learning models to estimate our confidence in predicting the control or diabetes status of placental transcriptome specimens with no available clinical metadata. CONCLUSION: Consistent with the distinct association of perinatal outcome risk, placentae from GDMA1, GDMA2, and T2DM-affected pregnancies harbor unique gene signatures that can be further distinguished by altered placental cellular subtypes and microarchitectural niches.

Quantitation and identification of microplastics accumulation in human placental specimens using pyrolysis gas chromatography mass spectrometry.

The exponential increase in global plastic usage has led to the emergence of nano- and microplastic (NMP) pollution as a pressing environmental issue due to its implications for human and other mammalian health. We have developed methodologies to extract solid materials from human tissue samples by saponification and ultracentrifugation, allowing for highly specific and quantitative analysis of plastics by pyrolysis-gas chromatography and mass spectrometry (Py-GC-MS). As a benchmark, placenta tissue samples were analyzed using fluorescence microscopy and automated particle count, which demonstrated the presence of >1-micron particles and fibers, but not nano-sized plastic particles. Analyses of the samples (n = 10) using attenuated total reflectance-Fourier transform infrared spectroscopy indicated presence of rayon, polystyrene, polyethylene, and unclassified plastic particles. By contrast, among 62 placenta samples, Py-GC-MS revealed that microplastics were present in all participants' placentae, with concentrations ranging widely from 6.5 to 685 µg NMPs per gram of placental tissue, averaging 126.8 ± 147.5 µg/g (mean±SD). Polyethylene was the most prevalent polymer, accounting for 54% of total NMPs and consistently found in nearly all samples (mean 68.8 ± 93.2 µg/g placenta). Polyvinyl chloride and nylon each represented approximately 10% of the NMPs by weight, with the remaining 26% of the composition represented by 9 other polymers. Together, these data demonstrate advancements in the unbiased quantitative resolution of Py-GC-MS applied to the identification and quantification of NMP species at the maternal-fetal interface. This method, paired with clinical metadata, will be pivotal to evaluating potential impacts of NMPs on adverse pregnancy outcomes.

Zika virus co-opts microRNA networks to persist in placental niches detected by spatial transcriptomics.

BACKGROUND: Zika virus congenital infection evades double-stranded RNA detection and may persist in the placenta for the duration of pregnancy without accompanying overt histopathologic inflammation. Understanding how viruses can persist and replicate in the placenta without causing overt cellular or tissue damage is fundamental to deciphering mechanisms of maternal-fetal vertical transmission. OBJECTIVE: Placenta-specific microRNAs are believed to be a tenet of viral resistance at the maternal-fetal interface. We aimed to test the hypothesis that the Zika virus functionally disrupts placental microRNAs, enabling viral persistence and fetal pathogenesis. STUDY DESIGN: To test this hypothesis, we used orthogonal approaches in human and murine experimental models. In primary human trophoblast cultures (n=5 donor placentae), we performed Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation to identify any significant alterations in the functional loading of microRNAs and their targets onto the RNA-induced silencing complex. Trophoblasts from same-donors were split and infected with a contemporary first-passage Zika virus strain HN16 (multiplicity of infection=1 plaque forming unit per cell) or mock infected. To functionally cross-validate microRNA-messenger RNA interactions, we compared our Argonaute high-throughput sequencing ultraviolet-crosslinking and immunoprecipitation results with an independent analysis of published bulk RNA-sequencing data from human placental disk specimens (n=3 subjects; Zika virus positive in first, second, or third trimester, CD45- cells sorted by flow cytometry) and compared it with uninfected controls (n=2 subjects). To investigate the importance of these microRNA and RNA interference networks in Zika virus pathogenesis, we used a gnotobiotic mouse model uniquely susceptible to the Zika virus. We evaluated if small-molecule enhancement of microRNA and RNA interference pathways with enoxacin influenced Zika virus pathogenesis (n=20 dams total yielding 187 fetal specimens). Lastly, placentae (n=14 total) from this mouse model were analyzed with Visium spatial transcriptomics (9743 spatial transcriptomes) to identify potential Zika virus-associated alterations in immune microenvironments. RESULTS: We found that Zika virus infection of primary human trophoblast cells led to an unexpected disruption of placental microRNA regulation networks. When compared with uninfected controls, Zika virus-infected placentae had significantly altered SLC12A8, SDK1, and VLDLR RNA-induced silencing complex loading and transcript levels (-22; adjusted P value <.05; Wald-test with false discovery rate correction q<0.05). In silico microRNA target analyses revealed that 26 of 119 transcripts (22%) in the transforming growth factor-ß signaling pathway were targeted by microRNAs that were found to be dysregulated following Zika virus infection in trophoblasts. In gnotobiotic mice, relative to mock controls, Zika virus-associated fetal pathogenesis included fetal growth restriction (P=.036) and viral persistence in placental tissue (P=.011). Moreover, spatial transcriptomics of murine placentae revealed that Zika virus-specific placental niches were defined by significant up-regulation of complement cascade components and coordinated changes in transforming growth factor-ß gene expression. Finally, treatment of Zika virus-infected mice with enoxacin abolished placental Zika virus persistence, rescued the associated fetal growth restriction, and the Zika virus-associated transcriptional changes in placental immune microenvironments were no longer observed. CONCLUSION: These results collectively suggest that (1) Zika virus infection and persistence is associated with functionally perturbed microRNA and RNA interference pathways specifically related to immune regulation in placental microenvironments and (2) enhancement of placental microRNA and RNA interference pathways in mice rescued Zika virus-associated pathogenesis, specifically persistence of viral transcripts in placental microenvironments and fetal growth restriction.

Gestational ozone inhalation elicits maternal cardiac dysfunction and transcriptional changes to placental pericytes and endothelial cells.

Ozone (O3) is a criteria air pollutant with the most frequent incidence of exceeding air quality standards. Inhalation of O3 is known to cause lung inflammation and consequent systemic health effects, including endothelial dysfunction. Epidemiologic data have shown that gestational exposure to air pollutants correlates with complications of pregnancy, including low birth weight, intrauterine growth deficiency, preeclampsia, and premature birth. Mechanisms underlying how air pollution may facilitate or exacerbate gestational complications remain poorly defined. The current study sought to uncover how gestational O3 exposure impacted maternal cardiovascular function, as well as the development of the placenta. Pregnant mice were exposed to 1PPM O3 or a sham filtered air (FA) exposure for 4 h on gestational day (GD) 10.5, and evaluated for cardiac function via echocardiography on GD18.5. Echocardiography revealed a significant reduction in maternal stroke volume and ejection fraction in maternally exposed dams. To examine the impact of maternal O3 exposure on the maternal-fetal interface, placentae were analyzed by single-cell RNA sequencing analysis. Mid-gestational O3 exposure led to significant differential expression of 4021 transcripts compared with controls, and pericytes displayed the greatest transcriptional modulation. Pathway analysis identified extracellular matrix organization to be significantly altered after the exposure, with the greatest modifications in trophoblasts, pericytes, and endothelial cells. This study provides insights into potential molecular processes during pregnancy that may be altered due to the inhalation of environmental toxicants.

SARS-CoV-2 niches in human placenta revealed by spatial transcriptomics.

BACKGROUND: Functional placental niches are presumed to spatially separate maternal-fetal antigens and restrict the vertical transmission of pathogens. We hypothesized a high-resolution map of placental transcription could provide direct evidence for niche microenvironments with unique functions and transcription profiles. METHODS: We utilized Visium Spatial Transcriptomics paired with H&E staining to generate 17,927 spatial transcriptomes. By integrating these spatial transcriptomes with 273,944 placental single-cell and single-nuclei transcriptomes, we generated an atlas composed of at least 22 subpopulations in the maternal decidua, fetal chorionic villi, and chorioamniotic membranes. FINDINGS: Comparisons of placentae from uninfected healthy controls (n = 4) with COVID-19 asymptomatic (n = 4) and symptomatic (n = 5) infected participants demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in syncytiotrophoblasts occurred in both the presence and the absence of maternal clinical disease. With spatial transcriptomics, we found that the limit of detection for SARS-CoV-2 was 1/7,000 cells, and placental niches without detectable viral transcripts were unperturbed. In contrast, niches with high SARS-CoV-2 transcript levels were associated with significant upregulation in pro-inflammatory cytokines and interferon-stimulated genes, altered metallopeptidase signaling (TIMP1), with coordinated shifts in macrophage polarization, histiocytic intervillositis, and perivillous fibrin deposition. Fetal sex differences in gene expression responses to SARS-CoV-2 were limited, with confirmed mapping limited to the maternal decidua in males. CONCLUSIONS: High-resolution placental transcriptomics with spatial resolution revealed dynamic responses to SARS-CoV-2 in coordinate microenvironments in the absence and presence of clinically evident disease. FUNDING: This work was supported by the NIH (R01HD091731 and T32-HD098069), NSF (2208903), the Burroughs Welcome Fund and the March of Dimes Preterm Birth Research Initiatives, and a Career Development Award from the American Society of Gene and Cell Therapy.

The third international hackathon for applying insights into large-scale genomic composition to use cases in a wide range of organisms.

In October 2021, 59 scientists from 14 countries and 13 U.S. states collaborated virtually in the Third Annual Baylor College of Medicine & DNANexus Structural Variation hackathon. The goal of the hackathon was to advance research on structural variants (SVs) by prototyping and iterating on open-source software. This led to nine hackathon projects focused on diverse genomics research interests, including various SV discovery and genotyping methods, SV sequence reconstruction, and clinically relevant structural variation, including SARS-CoV-2 variants. Repositories for the projects that participated in the hackathon are available at https://github.com/collaborativebioinformatics.

COVID-19 Pandemic and Infant Neurodevelopmental Impairment: A Systematic Review and Meta-analysis.

Importance: Primary studies proposed that aberrant maternal antiviral immunity and/or giving birth in quarantine, such as during the ongoing COVID-19 pandemic, may be associated with the risk of neurodevelopmental impairment (NDI) in offspring. Objectives: To evaluate the associations of birth and being raised during the COVID-19 pandemic with risk of NDI among infants and to assess the association of gestational exposure to SARS-CoV-2 with risk of NDI. Data Sources: PubMed, Web of Science, Scopus, Embase, and preprint servers were systematically searched from inception to March 25, 2022. Study Selection: Studies evaluating the neurodevelopment of infants born during the SARS-CoV-2 pandemic were included in this systematic review and meta-analysis. Studies using Ages and Stages Questionnaires, Third Edition (ASQ-3), were used for quantitative meta-analysis. Data Extraction and Synthesis: Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses, a random-effects model meta-analysis was used to pool the proportion and odds ratios (ORs) of overall NDI, as well as each developmental domain on ASQ-3 with the corresponding 95% CI. Main Outcomes and Measures: The primary outcome was the risk of overall NDI among infants screened during the pandemic vs prepandemic. The secondary outcome was the comparison of NDI by ASQ-3 domain among infants born to women with known gestational exposure to SARS-CoV-2 vs no exposure. Results: A total of 8 studies were included, including 21 419 infants (11 438 screened in pandemic and 9981 in prepandemic period). NDI was present in 330 of 8992 infants (7%; 95% CI, 4%-10%) screened during the COVID-19 pandemic from January 2020 to January 2021. Among the pandemic cohort, the prevalence of NDI among infants with gestational exposure to SARS-CoV-2 was 77 of 691 (12%; 95% CI, 6%-18%). Compared with the prepandemic cohort (2015-2019), the pandemic cohort was more likely to have communication impairment (OR, 1.70; 95% CI, 1.37-2.11; P < .001), without significant differences in other ASQ-3 domains (eg, gross motor, fine motor, personal-social, and problem-solving). In contrast, maternal SARS-CoV-2 infection was not associated with significant differences in any neurodevelopment domain in offspring, except for increasing the odds of fine motor impairment (OR, 3.46; 95% CI, 1.43-8.38; P < .001). Conclusions and Relevance: In this systematic review and meta-analysis examining the association between COVID-19 pandemic and the risk of NDI, findings suggest that overall neurodevelopment in the first year of life was not changed by either being born or raised during the SARS-CoV-2 pandemic or by gestational exposure to SARS-CoV-2. Interestingly, the first year of life during the COVID-19 pandemic, regardless of maternal infection, was significantly associated with the risk of communication delay among the offspring.

Placental Vascular and Inflammatory Findings from Pregnancies Diagnosed with Coronavirus Disease 2019: A Systematic Review and Meta-analysis.

We aimed to perform a meta-analysis of the literature concerning histopathologic findings in the placentas of women with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection during pregnancy. Searches for articles in English included PubMed, Web of Science, Google Scholar, and reference lists (up to April 2021). Studies presenting data on placental histopathology according to the Amsterdam Consensus Group criteria in SARS-CoV-2 positive and negative pregnancies were identified. Lesions were categorized into: maternal and fetal vascular malperfusion (MVM and FVM, respectively), acute placental inflammation with maternal and fetal inflammatory response (MIR and FIR, respectively), chronic inflammatory lesions (CILs), and increased perivillous fibrin deposition (PVFD). A total of 15 studies reporting on 19,025 placentas, n = 699 of which were derived from women who were identified as being infected with SARS-CoV-2 and 18,326 as SARS-CoV-2-negative controls, were eligible for analysis. No significant difference in incidence of MVM (odds ratio [OR]: 1.18, 95% confidence interval [CI]: 0.73-1.90), FVM (OR: 1.23, 95% CI: 0.63-2.42), MIR (OR: 0.66, 95% CI: 0.29-1.52) or FIR (OR: 0.85, 95% CI: 0.44-1.63), and CILs (OR: 0.97, 95% CI: 0.55-1.72) was found between placentae from gravida identified as being SARS-CoV-2 infected. However, placenta from gravida identified as being infected with SARS-CoV-2 were associated with significantly increased occurrence of PVFD (OR: 2.77, 95% CI: 1.06-7.27). After subgroup analyses based on clinical severity of COVID-19 infection, no significant difference was observed in terms of reported placental pathology between symptomatic or asymptomatic SARS-CoV-2 gravidae placenta. Current evidence based on the available literature suggests that the only pathologic finding in the placentae of women who are pregnant identified as having been infected with SARS-CoV-2 was an increased prevalence of PVFD. KEY POINTS: · No association between SARS-CoV-2 and maternal or fetal placental malperfusion.. · No association between SARS-CoV-2 and maternal or fetal inflammatory response.. · SARS-CoV-2 is associated with increased perivillous fibrin deposition in placenta..

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication.

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus in the family Pneumoviridae and genus Orthopneumovirus that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all S and T residues to alanine (A) and analyzed their effects on genome transcription and replication by using a minigenome system. We found that the mutation of eight residues resulted in minigenome activity significantly lower than that of wild-type (WT) P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (with T151A, S156A, T160A, or S23A), suggesting that RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four recombinant viruses rescued, rRSV-T160A caused a minor growth defect relative to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis. IMPORTANCE RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen.

Herpes Simplex Virus 1 MicroRNA miR-H8 Is Dispensable for Latency and Reactivation In Vivo.

The regulatory functions of 10 individual viral microRNAs (miRNAs) that are abundantly expressed from the herpes simplex virus 1 (HSV-1) latency-associated transcript (LAT) region remain largely unknown. Here, we focus on HSV-1 miRNA miR-H8, which is within the LAT 3p exon, antisense to the first intron of ICP0, and has previously been shown to target a host glycosylphosphatidylinositol (GPI)-anchoring pathway. However, the functions of this miRNA have not been assessed in the context of the viral genome during infection. Therefore, we constructed a recombinant virus lacking miR-H8 (17dmiR-H8) and compared it to the parental wild-type and rescue viruses to characterize phenotypic differences. In rabbit skin cells, 17dmiR-H8 exhibited only subtle reductions in viral yields. In contrast, we found significant decreases in both viral yields (8-fold) and DNA replication (9.9-fold) in murine neuroblastoma cells, while 17dmiR-H8 exhibited a 3.6-fold increase in DNA replication in differentiated human neuronal cells (Lund human mesencephalic [LUHMES] cells). These cell culture phenotypes suggested potential host- and/or neuron-specific roles for miR-H8 in acute viral replication. To assess whether miR-H8 plays a role in HSV latency or reactivation, we used a human in vitro reactivation model as well as mouse and rabbit reactivation models. In the LUHMES cell-induced reactivation model, there was no difference in viral yields at 48 h postreactivation. In the murine dorsal root ganglion explant and rabbit ocular adrenergic reactivation models, the deletion of miR-H8 had no detectable effect on genome loads during latency or reactivation. These results indicate that miR-H8 is dispensable for the establishment of HSV-1 latency and reactivation.IMPORTANCE Herpesviruses have a remarkable ability to sustain lifelong infections by evading host immune responses, establishing a latent reservoir, and maintaining the ability to reactivate the lytic cascade to transmit the virus to the next host. The HSV-1 latency-associated transcript region is known to regulate many aspects of HSV-1 latency and reactivation, although the mechanisms for these functions remain unknown. To this end, we characterize an HSV-1 recombinant containing a deletion of a LAT-encoded miRNA, miR-H8, and demonstrate that it plays no detectable role in the establishment of latency or reactivation in differentiated human neurons (LUHMES cells) and mouse and rabbit models. Therefore, this study allows us to exclude miR-H8 from phenotypes previously attributed to the LAT region. Elucidating the genetic elements of HSV-1 responsible for establishment, maintenance, and reactivation from latency may lead to novel strategies for combating persistent herpesvirus infections.

Deletion of Herpes Simplex Virus 1 MicroRNAs miR-H1 and miR-H6 Impairs Reactivation.

During all stages of infection, herpes simplex virus 1 (HSV-1) expresses viral microRNAs (miRNAs). There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. We constructed a recombinant virus lacking the sequences for miR-H1-5p and miR-H6-3p (17dmiR-H1/H6). The seed sequences for these miRNAs are antisense to each other and are transcribed from divergent noncoding RNAs in the latency-associated transcript (LAT) promoter region. Comparing phenotypes exhibited by the recombinant virus lacking these miRNAs to the wild type (17syn+), we found that during acute infection in cell culture, 17dmiR-H1/H6 exhibited a modest increase in viral yields. Analysis of pathogenesis in the mouse following footpad infection revealed a slight increase in virulence for 17dmiR-H1/H6 but no significant difference in the establishment or maintenance of latency. Strikingly, explant of latently infected dorsal root ganglia revealed a decreased and delayed reactivation phenotype. Further, 17dmiR-H1/H6 was severely impaired in epinephrine-induced reactivation in the rabbit ocular model. Finally, we demonstrated that deletion of miR-H1/H6 increased the accumulation of the LAT as well as several of the LAT region miRNAs. These results suggest that miR-H1/H6 plays an important role in facilitating efficient reactivation from latency.IMPORTANCE While HSV antivirals reduce the severity and duration of clinical disease in some individuals, there is no vaccine or cure. Therefore, understanding the mechanisms regulating latency and reactivation as a potential to elucidate targets for better therapeutics is important. There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. The present study focuses on two of the miRNAs (miR-H1/H6) that are encoded within the latency-associated transcript (LAT) region, a portion of the genome that has been associated with efficient reactivation. Here, we demonstrate that the deletion of the seed sequences of these miRNAs results in a severe reduction in reactivation of HSV-1 in the mouse and rabbit models. These results suggest a linkage between these miRNAs and reactivation.

HSV1 latent transcription and non-coding RNA: A critical retrospective.

Virologists have invested great effort into understanding how the herpes simplex viruses and their relatives are maintained dormant over the lifespan of their host while maintaining the poise to remobilize on sporadic occasions. Piece by piece, our field has defined the tissues in play (the sensory ganglia), the transcriptional units (the latency-associated transcripts), and the responsive genomic region (the long repeats of the viral genomes). With time, the observed complexity of these features has compounded, and the totality of viral factors regulating latency are less obvious. In this review, we compose a comprehensive picture of the viral genetic elements suspected to be relevant to herpes simplex virus 1 (HSV1) latent transcription by conducting a critical analysis of about three decades of research. We describe these studies, which largely involved mutational analysis of the notable latency-associated transcripts (LATs), and more recently a series of viral miRNAs. We also intend to draw attention to the many other less characterized non-coding RNAs, and perhaps coding RNAs, that may be important for consideration when trying to disentangle the multitude of phenotypes of the many genetic modifications introduced into recombinant HSV1 strains.

Exposure to D2-like dopamine receptor agonists inhibits swimming in Daphnia magna.

Daphnia are freshwater crustaceans that have been used for decades in ecotoxicology research. Despite the important role that Daphnia have played in environmental toxicology studies, very little is known about the neurobiology of Daphnia. Although many studies have investigated the swimming movements of these "water fleas", few studies have examined the underlying neurochemical basis for these movements. To characterize the locomotor effect of drugs in Daphnia, a two-dimensional video imaging tool was developed and animal tracking was performed with freely available software, CTRAX. Due to the central role that dopamine plays in the movement of animals, we sought to determine the role of dopamine receptor signaling in Daphnia movement by characterizing the effect of ten drugs that are agonists or antagonists of dopamine receptors. At 1, 2, and 6h of treatment with a 10µM drug, several dopamine receptor agonists with documented effects on the D2-like class of receptors decreased the movement. Further, we determined behavioral inhibition values (IC50) at 1h of treatment for (1R,3S)-1-(aminomethyl)-3-phenyl-3,4-dihydro-1H-isochromene-5,6-diol (A68930) to be 1.4µM and for bromocriptine to be 6.6µM. This study describes a new method to study Daphnia swimming and establishes this organism as a useful model for studies of dopaminergic signaling. Specifically, this study shows that a dopamine receptor signaling pathway, mediated by putative D2-like receptors, is involved in the control of Daphnia swimming behavior. Due to its ease of use and its rich motor program we propose that Daphnia should be considered for future studies of dopamine neuron toxicity and protection.