Sindbis virus usurps the cellular HUR protein to stabilize its transcripts and promote infections of mammalian and mosquito cells

2010 Summer.Includes bibliographic references (pages 174-208).Covers not scanned.Print version deaccessioned 2022.Members of the genus Alphavirus are recognized as significant human pathogens. Infection of vertebrate hosts often results in febrile illness and occasionally severe encephalitis. The archetypical alphavirus is Sindbis virus, which we have utilized in these studies. The genomic and subgenomic RNAs of Sindbis virus strongly resemble cellular mRNAs as they are capped at their 5’ ends and polyadenylated at their 3’ termini. These features allow the viral RNAs to act like cellular mRNAs and make them prime substrates for the cellular mRNA decay machinery. Sindbis virus RNAs are indeed subject to degradation by the cellular mRNA decay machinery in cell culture models of infection. Nevertheless, they decay by a mechanism that is different from the majority of cellular mRNAs as the decay of Sindbis virus transcripts is predominantly deadenylation-independent. As cellular mRNAs are often regulated by elements present in their 3’ untranslated regions (UTR), we hypothesized that these viral 3’UTR elements were functioning similarly to cellular mRNA stability elements resulting in the enhancement of viral infection. The primary goal of the research described in this dissertation was to characterize in mechanistic detail how the Sindbis virus 3’UTR represses deadenylation. To this end we used both cell free extracts and tissue culture systems to assay the effects of the viral 3’UTR on transcript stability. Interestingly, multiple elements were found to be independently repressing deadenylation in mosquito cytoplasmic extracts. Further examination revealed that a major stability determinant was the U-rich element (URE) observed in the 3’UTR of many alphaviruses. The ability to repress deadenylation in our cell free extract system was similarly observed with the UREs of Venezuelan equine, eastern equine, western equine and Semliki Forest viruses. Taken together, these data strongly assert that the repression of deadenylation via the URE is evolutionarily conserved. Prior to this study, the URE had no ascribed function. The repression of deadenylation imparted by the URE correlated with the binding of a cellular 38kDa factor. This 38kDa factor was determined to be the cellular HuR protein. Both the human and mosquito HuR proteins were found to bind with high affinity to the Sindbis virus 3’UTR. Reduction of cellular HuR protein levels using RNAi resulted in an increase in the rate of viral RNA decay. Furthermore, a significant decrease in the titer of progeny virus was observed. A similar effect on viral titer was observed when the predominant HuR binding site, the URE, was deleted from the viral 3’UTR. Taken together these observations identify a novel Alphavirus/ host interface that significantly impacts viral biology. Furthermore these studies have confirmed our hypothesis that the members of genus Alphavirus have indeed evolved RNA stability elements that resemble cellular mRNA stability elements for the purpose of enhancing viral infection. Furthermore these studies identify a potential therapeutic anti-viral target - the cellular HuR protein

Combining community wastewater genomic surveillance with state clinical surveillance: A framework for SARS-CoV-2 public health practice

Study objective: To garner a framework for combining community wastewater surveillance with state clinical surveillance that influence confirmation of SARS-CoV-2 variants within the community, and recommend how the flow of such research evidence could be expanded and employed for public health response. Design, setting, and participants: This work involved analyzing wastewater samples collected weekly from 17 geographically resolved locations in Louisville/Jefferson County, Kentucky from February 10 to November 29, 2021. Genomic surveillance and RT-qPCR platforms were used as screening to identify SARS-CoV-2 in wastewater, and state clinical surveillance was used for confirmation. Main results: The results demonstrate increased epidemiological value of combining community wastewater genomic surveillance and RT-qPCR with conventional case auditing methods. The spatial scale and temporal frequency of wastewater sampling provides promising sensitivity and specificity to be useful to gain public health screening insights about community emergence, seeding, and spread. Conclusions: Better national surveillance systems are needed for future pathogens and variants, and wastewater-based genomic surveillance represents opportune coupling. This paper presents current evidence that complementary wastewater and clinical testing is enhanced cost-effectively when linked; making a strong case for a joint public health framework. The findings suggest significant potential for rapid progress to be made in extending this work to consider pathogens of interest as a whole within wastewater, which could be examined in either a targeted fashion as we currently do with SARS-CoV-2 or in terms of a global monitoring of all pathogens found, and developing evidence based public health practice to best support community health

Intranasal Antiviral Drug Delivery and Coronavirus Disease 2019 (COVID-19): A State of the Art Review

Objective To provide a state of the art review of intranasal antiviral drug delivery and to discuss current applications, adverse reactions, and future considerations in the management of coronavirus disease 2019 (COVID-19). Data Sources PubMed, Embase, and Clinicaltrials.gov search engines. Review Methods A structured search of the current literature was performed of dates up to and including April 2020. Search terms were queried as related to topics of antiviral agents and intranasal applications. A series of video conferences was convened among experts in otolaryngology, infectious diseases, public health, pharmacology, and virology to review the literature and discuss relevant findings. Conclusions Intranasal drug delivery for antiviral agents has been studied for many years. Several agents have broad-spectrum antiviral activity, but they still require human safety and efficacy trials prior to implementation. Intranasal drug delivery has potential relevance for future clinical trials in the settings of disease spread prevention and treatment of SARS-CoV-2 and other viral diseases

The rapid assessment of aggregated wastewater samples for genomic surveillance of sars-cov-2 on a city-wide scale

Throughout the course of the ongoing SARS-CoV-2 pandemic there has been a need for approaches that enable rapid monitoring of public health using an unbiased and minimally invasive means. A major way this has been accomplished is through the regular assessment of wastewater samples by qRT-PCR to detect the prevalence of viral nucleic acid with respect to time and location. Further expansion of SARS-CoV-2 wastewater monitoring efforts to include the detection of variants of interest/concern through next-generation sequencing has enhanced the understanding of the SARS-CoV-2 outbreak. In this report, we detail the results of a collaborative effort between public health and metropolitan wastewater management authorities and the University of Louisville to monitor the SARS-CoV-2 pandemic through the monitoring of aggregate wastewater samples over a period of 28 weeks. Through the use of next-generation sequencing approaches the polymorphism signatures of Variants of Concern / Interest were evaluated to determine the likelihood of their prevalence within the community on the basis of their relative dominance within sequence datasets. Our data indicate that wastewater monitoring of water quality treatment centers and smaller neighbor-hood-scale catchment areas is a viable means by which the prevalence and genetic variation of SARS-CoV-2 within a metropolitan community of approximately one million individuals may be monitored, as our efforts detected the introduction and emergence of variants of concern in the city of Louisville. Importantly, these efforts confirm that regional emergence and spread of variants of interest/concern may be detected as readily in aggregate wastewater samples as compared to the individual wastewater sheds. Furthermore, the information gained from these efforts enabled targeted public health efforts including increased outreach to at-risk communities and the deployment of mobile or community-focused vaccination campaigns

Palomar Gattini-IR: Survey overview, data processing system, on-sky performance and first results

Palomar Gattini-IR is a new wide-field, near-infrared (NIR) robotic time domain survey operating at Palomar Observatory. Using a 30 cm telescope mounted with a H2RG detector, Gattini-IR achieves a field of view (FOV) of 25 sq. deg. with a pixel scale of 8.”7 in J-band. Here, we describe the system design, survey operations, data processing system and on-sky performance of Palomar Gattini-IR. As a part of the nominal survey, Gattini-IR scans ≈7500 square degrees of the sky every night to a median 5σ depth of 15.7 AB mag outside the Galactic plane. The survey covers ≈15,000 square degrees of the sky visible from Palomar with a median cadence of 2 days. A real-time data processing system produces stacked science images from dithered raw images taken on sky, together with point-spread function (PSF)-fit source catalogs and transient candidates identified from subtractions within a median delay of ≈4 hr from the time of observation. The calibrated data products achieve an astrometric accuracy (rms) of ≈0.”7 with respect to Gaia DR2 for sources with signal-to-noise ratio > 10, and better than ≈0.”35 for sources brighter than ≈12 Vega mag. The photometric accuracy (rms) achieved in the PSF-fit source catalogs is better than ≈3% for sources brighter than ≈12 Vega mag and fainter than the saturation magnitude of ≈8.5 Vega mag, as calibrated against the Two Micron All Sky Survey catalog. The detection efficiency of transient candidates injected into the images is better than 90% for sources brighter than the 5σ limiting magnitude. The photometric recovery precision of injected sources is 3% for sources brighter than 13 mag, and the astrometric recovery rms is ≈0.”9. Reference images generated by stacking several field visits achieve depths of ≳16.5 AB mag over 60% of the sky, while it is limited by confusion in the Galactic plane. With a FOV ≈40× larger than any other existing NIR imaging instrument, Gattini-IR is probing the reddest and dustiest transients in the local universe such as dust obscured supernovae in nearby galaxies, novae behind large columns of extinction within the galaxy, reddened microlensing events in the Galactic plane and variability from cool and dust obscured stars. We present results from transients and variables identified since the start of the commissioning period

CfA3: 185 Type Ia Supernova Light Curves from the CfA

We present multi-band photometry of 185 type-Ia supernovae (SN Ia), with over 11500 observations. These were acquired between 2001 and 2008 at the F. L. Whipple Observatory of the Harvard-Smithsonian Center for Astrophysics (CfA). This sample contains the largest number of homogeneously-observed and reduced nearby SN Ia (z < 0.08) published to date. It more than doubles the nearby sample, bringing SN Ia cosmology to the point where systematic uncertainties dominate. Our natural system photometry has a precision of 0.02 mag or better in BVRIr'i' and roughly 0.04 mag in U for points brighter than 17.5 mag. We also estimate a systematic uncertainty of 0.03 mag in our SN Ia standard system BVRIr'i' photometry and 0.07 mag for U. Comparisons of our standard system photometry with published SN Ia light curves and comparison stars, where available for the same SN, reveal agreement at the level of a few hundredths mag in most cases. We find that 1991bg-like SN Ia are sufficiently distinct from other SN Ia in their color and light-curve-shape/luminosity relation that they should be treated separately in light-curve/distance fitter training samples. The CfA3 sample will contribute to the development of better light-curve/distance fitters, particularly in the few dozen cases where near-infrared photometry has been obtained and, together, can help disentangle host-galaxy reddening from intrinsic supernova color, reducing the systematic uncertainty in SN Ia distances due to dust.Comment: Accepted to the Astrophysical Journal. Minor changes from last version. Light curves, comparison star photometry, and passband tables are available at http://www.cfa.harvard.edu/supernova/CfA3

Comparative analyses of alphaviral RNA:Protein complexes reveals conserved host-pathogen interactions.

The identification of host / pathogen interactions is essential to both understanding the molecular biology of infection and developing rational intervention strategies to overcome disease. Alphaviruses, such as Sindbis virus, Chikungunya virus, and Venezuelan Equine Encephalitis virus are medically relevant positive-sense RNA viruses. As such, they must interface with the host machinery to complete their infectious lifecycles. Nonetheless, exhaustive RNA:Protein interaction discovery approaches have not been reported for any alphavirus species. Thus, the breadth and evolutionary conservation of host interactions on alphaviral RNA function remains a critical gap in the field. Herein we describe the application of the Cross-Link Assisted mRNP Purification (CLAMP) strategy to identify conserved alphaviral interactions. Through comparative analyses, conserved alphaviral host / pathogen interactions were identified. Approximately 100 unique host proteins were identified as a result of these analyses. Ontological assessments reveal enriched Molecular Functions and Biological Processes relevant to alphaviral infection. Specifically, as anticipated, Poly(A) RNA Binding proteins are significantly enriched in virus specific CLAMP data sets. Moreover, host proteins involved in the regulation of mRNA stability, proteasome mediated degradation, and a number of 14-3-3 proteins were identified. Importantly, these data expand the understanding of alphaviral host / pathogen interactions by identifying conserved interactants