SARS-CoV-2 Variant Tracking and Mitigation During In-Person Learning at a Midwestern University in the 2020-2021 School Year.

Importance: The COVID-19 pandemic led many higher education institutions to close campuses during the 2020-2021 academic year. As campuses prepared for a return to in-person education, many institutions were mandating vaccines for students and considering the same for faculty and staff. Objective: To determine the association between vaccination coverage and the levels and spread of SARS-CoV-2, even in the presence of highly-transmissible variants and congregate living, at a midsized university in the US. Design, Setting, and Participants: This case series was conducted at a midsized Midwestern university during the spring 2021 semester. The university developed a saliva-based surveillance program capable of high-throughput SARS-CoV-2 polymerase chain reaction testing and genomic sequencing with the capacity to deliver results in less than 24 hours. On April 7, 2021, the university announced a vaccine requirement for all students for the fall 2021 semester and announced the same requirement for faculty and staff on May 20, 2021. The university hosted an onsite mass vaccination clinic using the 2-dose Pfizer-BioNTech vaccine during April 8 to 15 and April 29 to May 6, 2021. Data were analyzed for 14 894 individuals from the university population who were tested for COVID-19 on campus from January 6 to May 20, 2021. Main Outcomes and Measures: Positive SARS-CoV-2 diagnosis was confirmed by quantitative reverse transcription-polymerase chain reaction of saliva specimens, and variant identity was assessed by quantitative reverse transcription-polymerase chain reaction and next-generation sequencing of viral genomes. Results: Between January 6 and May 20, 2021, the university conducted 196 185 COVID-19 tests for 14 894 individuals and identified 1603 positive cases. Within those positive cases, 950 individuals (59.3%) were male, 644 (40.2%) were female, 1426 (89.0%) were students, and 1265 (78.9%) were aged 17 to 22 years. Among the 1603 positive cases, 687 were identified via polymerase chain reaction of saliva specimens. The Alpha (B.1.1.7) variant constituted 218 of the 446 total positives sequenced (48.9%). By May 20, 2021, 10 068 of 11 091 students (90.8%), 814 of 883 faculty (92.2%), and 2081 of 2890 staff (72.0%) were vaccinated. The 7-day rolling average of positive cases peaked at 37 cases on February 17 but declined to zero by May 14, 2021. The 7-day moving average of positive cases was inversely associated with cumulative vaccination coverage, with a statistically significant Pearson correlation coefficient of -0.57 (95% CI, -0.68 to -0.44). Conclusions and Relevance: This case series study elucidated the association of a robust vaccination program with a statistically significant decrease in positive COVID-19 cases among the study population even in the presence of highly transmissible variants and congregate living.

High-throughput cis-regulatory element discovery in the vector mosquito Aedes aegypti.

BACKGROUND: Despite substantial progress in mosquito genomic and genetic research, few cis-regulatory elements (CREs), DNA sequences that control gene expression, have been identified in mosquitoes or other non-model insects. Formaldehyde-assisted isolation of regulatory elements paired with DNA sequencing, FAIRE-seq, is emerging as a powerful new high-throughput tool for global CRE discovery. FAIRE results in the preferential recovery of open chromatin DNA fragments that are not bound by nucleosomes, an evolutionarily conserved indicator of regulatory activity, which are then sequenced. Despite the power of the approach, FAIRE-seq has not yet been applied to the study of non-model insects. In this investigation, we utilized FAIRE-seq to profile open chromatin and identify likely regulatory elements throughout the genome of the human disease vector mosquito Aedes aegypti. We then assessed genetic variation in the regulatory elements of dengue virus susceptible (Moyo-S) and refractory (Moyo-R) mosquito strains. RESULTS: Analysis of sequence data obtained through next generation sequencing of FAIRE DNA isolated from A. aegypti embryos revealed >121,000 FAIRE peaks (FPs), many of which clustered in the 1 kb 5' upstream flanking regions of genes known to be expressed at this stage. As expected, known transcription factor consensus binding sites were enriched in the FPs, and of these FoxA1, Hunchback, Gfi, Klf4, MYB/ph3 and Sox9 are most predominant. All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays. Of the >13,000 single nucleotide polymorphisms (SNPs) recently identified in dengue virus-susceptible and refractory mosquito strains, 3365 were found to map to FPs. CONCLUSION: FAIRE-seq analysis of open chromatin in A. aegypti permitted genome-wide discovery of CREs. The results of this investigation indicate that FAIRE-seq is a powerful tool for identification of regulatory DNA in the genomes of non-model organisms, including human disease vector mosquitoes.

Requirement for commissureless2 function during dipteran insect nerve cord development.

BACKGROUND: In Drosophila melanogaster, commissureless (comm) function is required for proper nerve cord development. Although comm orthologs have not been identified outside of Drosophila species, some insects possess orthologs of Drosophila comm2, which may also regulate embryonic nerve cord development. Here, this hypothesis is explored through characterization of comm2 genes in two disease vector mosquitoes. RESULTS: Culex quinquefasciatus (West Nile and lymphatic filiariasis vector) has three comm2 genes that are expressed in the developing nerve cord. Aedes aegypti (dengue and yellow fever vector) has a single comm2 gene that is expressed in commissural neurons projecting axons toward the midline. Loss of comm2 function in both A. aegypti and D. melanogaster was found to result in loss of commissure defects that phenocopy the frazzled (fra) loss of function phenotypes observed in both species. Loss of fra function in either insect was found to result in decreased comm2 transcript levels during nerve cord development. CONCLUSIONS: The results of this investigation suggest that Fra down-regulates repulsion in precrossing commissural axons by regulating comm2 levels in both A. aegypti and D. melanogaster, both of which require Comm2 function for proper nerve cord development.

Comparative genomic analysis of Drosophila melanogaster and vector mosquito developmental genes.

Genome sequencing projects have presented the opportunity for analysis of developmental genes in three vector mosquito species: Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A comparative genomic analysis of developmental genes in Drosophila melanogaster and these three important vectors of human disease was performed in this investigation. While the study was comprehensive, special emphasis centered on genes that 1) are components of developmental signaling pathways, 2) regulate fundamental developmental processes, 3) are critical for the development of tissues of vector importance, 4) function in developmental processes known to have diverged within insects, and 5) encode microRNAs (miRNAs) that regulate developmental transcripts in Drosophila. While most fruit fly developmental genes are conserved in the three vector mosquito species, several genes known to be critical for Drosophila development were not identified in one or more mosquito genomes. In other cases, mosquito lineage-specific gene gains with respect to D. melanogaster were noted. Sequence analyses also revealed that numerous repetitive sequences are a common structural feature of Drosophila and mosquito developmental genes. Finally, analysis of predicted miRNA binding sites in fruit fly and mosquito developmental genes suggests that the repertoire of developmental genes targeted by miRNAs is species-specific. The results of this study provide insight into the evolution of developmental genes and processes in dipterans and other arthropods, serve as a resource for those pursuing analysis of mosquito development, and will promote the design and refinement of functional analysis experiments.

The Drosophila Netrin receptor frazzled/DCC functions as an invasive tumor suppressor.

BACKGROUND: Loss of heterozygosity at 18q, which includes the Deleted in Colorectal Cancer (DCC) gene, has been linked to many human cancers. However, it is unclear if loss of DCC is the specific underlying cause of these cancers. The Drosophila imaginal discs are excellent systems in which to study DCC function, as it is possible to model human tumors through the generation of somatic clones of cells bearing multiple genetic lesions. Here, these attributes of the fly system were utilized to investigate the potential tumor suppressing functions of the Drosophila DCC homologue frazzled (fra) during eye-antennal disc development. RESULTS: Most fra loss of function clones are eliminated during development. However, when mutant clone cells generated in the developing eye were rescued from death, partially differentiated eye cells were found outside of the normal eye field, and in extreme cases distant sites of the body. Characterization of these cells during development indicates that fra mutant cells display characteristics of invasive tumor cells, including increased levels of phospho-ERK, phospho-JNK, and Mmp-1, changes in cadherin expression, remodeling of the actin cytoskeleton, and loss of polarity. Mutation of fra promotes basement membrane degradation and invasion which are repressed by inhibition of Rho1 signaling. Although inhibition of JNK signaling blocks invasive phenotypes in some metastatic cancer models in flies, blocking JNK signaling inhibits fra mutant cell death, thereby enhancing the fra mutant phenotype. CONCLUSIONS: The results of this investigation provide the first direct link between point mutations in fra/DCC and metastatic phenotypes in an animal model and suggest that Fra functions as an invasive tumor suppressor during Drosophila development.

Aedes aegypti: an emerging model for vector mosquito development.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although the Ae. aegypti genome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development in Ae. aegypti or other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes during Ae. aegypti development. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.