Transcriptional control of a metabolic switch regulating cellular methylation reactions is part of a common response to stress in divergent bee species.

Recent global declines in bee health have elevated the need for a more complete understanding of the cellular stress mechanisms employed by diverse bee species. We recently uncovered the biomarker lethal (2) essential for life [l(2)efl] genes as part of a shared transcriptional program in response to a number of cell stressors in the western honey bee (Apis mellifera). Here, we describe another shared stress-responsive gene, glycine N-methyltransferase (Gnmt), which is known as a key metabolic switch controlling cellular methylation reactions. We observed Gnmt induction by both abiotic and biotic stressors. We also found increased levels of the GNMT reaction product sarcosine in the midgut after stress, linking metabolic changes with the observed changes in gene regulation. Prior to this study, Gnmt upregulation had not been associated with cellular stress responses in other organisms. To determine whether this novel stress-responsive gene would behave similarly in other bee species, we first characterized the cellular response to endoplasmic reticulum (ER) stress in lab-reared adults of the solitary alfalfa leafcutting bee (Megachile rotundata) and compared this with age-matched honey bees. The novel stress gene Gnmt was induced in addition to a number of canonical gene targets induced in both bee species upon unfolded protein response (UPR) activation, suggesting that stress-induced regulation of cellular methylation reactions is a common feature of bees. Therefore, this study suggests that the honey bee can serve as an important model for bee biology more broadly, although studies on diverse bee species will be required to fully understand global declines in bee populations.

La Crosse virus reassortants highlight genomic determinants of infection and pathogenesis.

The genomic determinants that contribute to orthobunyavirus infection and pathogenesis are not well-defined. In this study, we harnessed the process of reassortment to understand which viral factors drive change in the replication and pathogenesis of La Crosse virus (LACV). We systematically reassorted the genomic segments of two genetically similar Lineage I LACV isolates into six unique reassortants. Despite the parental isolates having high levels of RNA and protein consensus, the reassortants demonstrate how minimal changes in RNA and protein structure can have significant changes in viral growth and reproduction in vitro in mammalian and insect models. We observed that swapping the S segment between isolates led to differences in replication and assembly resulting in one non-rescuable reassortant and one viable reassortant that exhibited an increase in viral growth dynamics. Switching the M segment led to changes in viral plaque phenotype and growth kinetics. L segment reassortants similarly differed in changes in viral growth dynamics. We further explored the M segment reassortants in a neonate mouse model and observed a role for the M segment in neuroinflammation and virulence. Through reassortment of the La Crosse virus genomic segments, we are able to further understand how genomic determinants of infection and pathogenesis operate in orthobunyaviruses. Future investigations will focus on identifying the specific molecular elements that govern the observed phenotypes in vitro and in vivo . Importance: La Crosse virus is the leading cause of pediatric arboviral encephalitis in the United States, yet it is largely unknown how each of the three genomic segments contribute to pathogenesis and disease. Our study utilizes genomic reassortment between two similar Lineage I LACV isolates to understand genomic determinants for differences in infection and pathogenesis phenotypes in vitro and in vivo. By identifying roles for each segment in observed outcomes, we are able to plan further studies for molecular characterization of these phenotypes. Additionally, it is imperative to continue to characterize orthobunyavirus function since climate change will expand the range and prevalence of arthropod-borne diseases such as LACV in the United States.

Building-Level Detection Threshold of SARS-CoV-2 in Wastewater.

We established wastewater surveillance of SARS-CoV-2 in a small, residential, urban college as part of an integrated public health response during the COVID-19 pandemic. Students returned to campus in spring 2021. During the semester, students were required to perform nasal PCR tests twice weekly. At the same time, wastewater monitoring was established in 3 campus dormitory buildings. Two were dedicated dormitories with populations of 188 and 138 students; 1 was an isolation building where students were moved within 2 h of receiving positive test results. Analysis of wastewater from isolation indicated that the amount of viral shedding was highly variable and that viral concentration could not be used to estimate the number of cases at the building level. However, rapid movement of students to isolation enabled determination of predictive power, specificity, and sensitivity from instances in which generally one positive case at a time occurred in a building. Our assay yields effective results with an ~60% positive predictive power, ~90% negative predictive power, and ~90% specificity. Sensitivity, however, is low at ~40%. Detection is improved in the few instances of 2 simultaneous positive cases, with sensitivity of 1 case versus 2 cases increasing from ~20% to 100%. We also measured the appearance of a variant of concern on campus and noted a similarity in timeline with increased prevalence in surrounding New York City. Monitoring SARS-CoV-2 in the sewage outflow of individual buildings can be used with a realistic goal of containing outbreak clusters but not necessarily single cases. IMPORTANCE Diagnostic testing of sewage can detect levels of circulating viruses to help inform public health. Wastewater-based epidemiology has been particularly active during the COVID-19 pandemic to measure the prevalence of SARS-CoV-2. Understanding the technical limitations of diagnostic testing for individual buildings would help inform future surveillance programs. We report our diagnostic and clinical data monitoring of buildings on a college campus in New York City during the spring 2021 semester. Frequent nasal testing, mitigation measures, and public health protocols provided a context in which to study the effectiveness of wastewater-based epidemiology. Our efforts could not consistently detect individual positive COVID-19 cases, but sensitivity is significantly improved in detecting two simultaneous cases. We therefore contend that wastewater surveillance may be more practically suited for the mitigation of outbreak clusters.

Honey bee sHSP are responsive to diverse proteostatic stresses and potentially promising biomarkers of honey bee stress.

The pollination services provided by the honey bee are critical in both natural and agricultural ecosystems. Honey bee colonies in the United States have suffered from an increased rate of die-off in recent years, stemming from a complex set of interacting stresses that remain poorly described. Defining specific common cellular processes and cellular stress responses impacted by multiple stressors represent a key step in understanding these synergies. Proteotoxic stresses negatively impact protein synthesis, folding, and degradation. Diverse proteotoxic stresses induce expression of genes encoding small heat shock proteins (sHSP) of the expanded lethal (2) essential for life (l(2)efl) gene family. In addition to upregulation by the Integrated Stress Response (ISR), the Heat Shock Response (HSR), and the Oxidative Stress Response (OSR), our data provide first evidence that sHSP genes are upregulated by the Unfolded Protein Response (UPR). As these genes appear to be part of a core stress response that could serve as a useful biomarker for cellular stress in honey bees, we designed and tested an RT-LAMP assay to detect increased l(2)efl gene expression in response to heat-stress. While this assay provides a powerful proof of principle, further work will be necessary to link changes in sHSP gene expression to colony-level outcomes, to adapt our preliminary assay into a Point of Care Testing (POCT) assay appropriate for use as a diagnostic tool for use in the field, and to couple assay results to management recommendations.

Rationally repurposed nitroxoline inhibits preclinical models of Epstein-Barr virus-associated lymphoproliferation.

Repurposing of currently used drugs for new indications benefits from known experience with those agents. Rational repurposing can be achieved when newly uncovered molecular activities are leveraged against diseases that utilize those mechanisms. Nitroxoline is an antibiotic with metal-chelating activity used to treat urinary tract infections. This small molecule also inhibits the function of bromodomain and extraterminal (BET) proteins that regulate oncogene expression in cancer. Lymphoproliferation driven by the Epstein-Barr virus (EBV) depends on these same proteins. We therefore tested the efficacy of nitroxoline against cell culture and small animal models of EBV-associated lymphoproliferation. Nitroxoline indeed reduces cell and tumor growth. Nitroxoline also acts faster than the prototype BET inhibitor JQ1. We suggest that this rational repurposing may hold translational promise.

Accessible and Validated Processing of SARS-CoV-2 from Wastewater.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), is shed into wastewater. Accessible methods are necessary for processing samples in a myriad of contexts. We optimized a protocol for extracting viral RNA for downstream experiments. Our pipeline was validated with SARS-CoV-2 itself as a matrix recovery and quantitative measurement control.

Widespread Traces of Lytic Kaposi Sarcoma-Associated Herpesvirus in Primary Effusion Lymphoma at Single-Cell Resolution.

Cancer cells of primary effusion lymphoma (PEL) often contain both Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). We measured the interplay of human, KSHV, and EBV transcription in a cell culture model of PEL using single-cell RNA sequencing. The data detect widespread trace expression of lytic KSHV genes.

The IRE1 pathway regulates honey bee Unfolded Protein Response gene expression.

Our molecular understanding of honey bee cellular stress responses is incomplete. Previously, we sought to identify and began functional characterization of the components of the Unfolded Protein Response (UPR) in honey bees. We observed that UPR stimulation resulted in induction of target genes upon IRE1 pathway activation, as assessed by splicing of Xbp1 mRNA. However, we were not able to determine the relative role of the various UPR pathways in gene activation. Our understanding of honey bee signal transduction and transcriptional regulation has been hampered by a lack of tools. After using RNA-seq to expand the known UPR targets in the honey bee, we used the Drosophila melanogaster S2 cell line and honey bee trans and cis elements to investigate the role of the IRE1 pathway in the transcriptional activation of one of these targets, the honey bee Hsc70-3 gene. Using a luciferase reporter, we show that honey bee Hsc70 promoter activity is inducible by UPR activation. In addition, we show that this activation is IRE1-dependent and relies on specific cis regulatory elements. Experiments using exogenous honey bee or fruit fly XBP1S proteins demonstrate that both factors can activate the Hsc70-3 promoter and further support a role for the IRE1 pathway in control of Hsc70-3 expression in the honey bee. By providing foundational knowledge about the UPR in the honey bee and demonstrating the usefulness of a heterologous cell line for molecular characterization of honey bee pathways, this work stands to improve our understanding of this critical species.