COVID-19 trends at the University of Tennessee: predictive insights from raw sewage SARS-CoV-2 detection and evaluation and PMMoV as an indicator for human waste.

Wastewater-based epidemiology (WBE) has become a valuable tool for monitoring the prevalence of SARS-CoV-2 on university campuses. However, concerns about effectiveness of raw sewage as a COVID-19 early warning system still exist, and it's not clear how useful normalization by simultaneous comparison of Pepper Mild Mottle Virus (PMMoV) is in addressing variations resulting from fecal discharge dilution. This study aims to contribute insights into these aspects by conducting an academic-year field trial at the student residences on the University of Tennessee, Knoxville campus, raw sewage. This was done to investigate the correlations between SARS-CoV-2 RNA load, both with and without PMMoV normalization, and various parameters, including active COVID-19 cases, self-isolations, and their combination among all student residents. Significant positive correlations between SARS-CoV-2 RNA load a week prior, during the monitoring week, and the subsequent week with active cases. Despite these correlations, normalization by PMMoV does not enhance these associations. These findings suggest the potential utility of SARS-CoV-2 RNA load as an early warning indicator and provide valuable insights into the application and limitations of WBE for COVID-19 surveillance specifically within the context of raw sewage on university campuses.

Evaluating various composite sampling modes for detecting pathogenic SARS-CoV-2 virus in raw sewage.

Inadequate sampling approaches to wastewater analyses can introduce biases, leading to inaccurate results such as false negatives and significant over- or underestimation of average daily viral concentrations, due to the sporadic nature of viral input. To address this challenge, we conducted a field trial within the University of Tennessee residence halls, employing different composite sampling modes that encompassed different time intervals (1 h, 2 h, 4 h, 6 h, and 24 h) across various time windows (morning, afternoon, evening, and late-night). Our primary objective was to identify the optimal approach for generating representative composite samples of SARS-CoV-2 from raw wastewater. Utilizing reverse transcription-quantitative polymerase chain reaction, we quantified the levels of SARS-CoV-2 RNA and pepper mild mottle virus (PMMoV) RNA in raw sewage. Our findings consistently demonstrated that PMMoV RNA, an indicator virus of human fecal contamination in water environment, exhibited higher abundance and lower variability compared to pathogenic SARS-CoV-2 RNA. Significantly, both SARS-CoV-2 and PMMoV RNA exhibited greater variability in 1 h individual composite samples throughout the entire sampling period, contrasting with the stability observed in other time-based composite samples. Through a comprehensive analysis of various composite sampling modes using the Quade Nonparametric ANCOVA test with date, PMMoV concentration and site as covariates, we concluded that employing a composite sampler during a focused 6 h morning window for pathogenic SARS-CoV-2 RNA is a pragmatic and cost-effective strategy for achieving representative composite samples within a single day in wastewater-based epidemiology applications. This method has the potential to significantly enhance the accuracy and reliability of data collected at the community level, thereby contributing to more informed public health decision-making during a pandemic.

Batrachochytrium salamandrivorans Can Devour More than Salamanders.

Batrachochytrium salamandrivorans is an emerging fungus that is causing salamander declines in Europe. We evaluated whether an invasive frog species (Cuban treefrog, Osteopilus septentrionalis) that is found in international trade could be an asymptomatic carrier when exposed to zoospore doses known to infect salamanders. We discovered that Cuban treefrogs could be infected with B. salamandrivorans and, surprisingly, that chytridiomycosis developed in animals at the two highest zoospore doses. To fulfill Koch's postulates, we isolated B. salamandrivorans from infected frogs, exposed eastern newts (Notophthalmus viridescens) to the isolate, and verified infection and disease by histopathology. This experiment represents the first documentation of B. salamandrivorans chytridiomycosis in a frog species and substantially expands the conservation threat and possible mobilization of this pathogen in trade.

Genomic Diversity of Type B3 Bacteriophages of Caulobacter crescentus.

The genomes of the type B3 bacteriophages that infect Caulobacter crescentus are among the largest phage genomes thus far deposited into GenBank with sizes over 200 kb. In this study, we introduce six new bacteriophage genomes which were obtained from phage collected from various water systems in the southeastern United States and from tropical locations across the globe. A comparative analysis of the 12 available genomes revealed a "core genome" which accounts for roughly 1/3 of these bacteriophage genomes and is predominately localized to the head, tail, and lysis gene regions. Despite being isolated from geographically distinct locations, the genomes of these bacteriophages are highly conserved in both genome sequence and gene order. We also identified the insertions, deletions, translocations, and horizontal gene transfer events which are responsible for the genomic diversity of this group of bacteriophages and demonstrated that these changes are not consistent with the idea that modular reassortment of genomes occurs in this group of bacteriophages.

D1398G Variant of MET Is Associated with Impaired Signaling of Hepatocyte Growth Factor in Alveolar Epithelial Cells and Lung Fibroblasts.

Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated "M10," with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFß-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor (CTGF, CCN2) and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients.

The Caulobacter crescentus transducing phage Cr30 is a unique member of the T4-like family of myophages.

Bacteriophage Cr30 has proven useful for the transduction of Caulobacter crescentus. Nucleotide sequencing of Cr30 DNA revealed that the Cr30 genome consists of 155,997 bp of DNA that codes for 287 proteins and five tRNAs. In contrast to the 67 % GC content of the host genome, the GC content of the Cr30 genome is only 38 %. This lower GC content causes both the codon usage pattern and the amino acid composition of the Cr30 proteins to be quite different from those of the host bacteria. As a consequence, the Cr30 mRNAs probably are translated at a rate that is slower than the normal rate for host mRNAs. A phylogenetic comparison of the genome indicates that Cr30 is a member of the T4-like family that is most closely related to a new group of T-like phages exemplified by фM12.

A comparison of the Caulobacter NA1000 and K31 genomes reveals extensive genome rearrangements and differences in metabolic potential.

The genus Caulobacter is found in a variety of habitats and is known for its ability to thrive in low-nutrient conditions. K31 is a novel Caulobacter isolate that has the ability to tolerate copper and chlorophenols, and can grow at 4 ° C with a doubling time of 40 h. K31 contains a 5.5 Mb chromosome that codes for more than 5500 proteins and two large plasmids (234 and 178 kb) that code for 438 additional proteins. A comparison of the K31 and the Caulobacter crescentus NA1000 genomes revealed extensive rearrangements of gene order, suggesting that the genomes had been randomly scrambled. However, a careful analysis revealed that the distance from the origin of replication was conserved for the majority of the genes and that many of the rearrangements involved inversions that included the origin of replication. On a finer scale, numerous small indels were observed. K31 proteins involved in essential functions shared 80-95% amino acid sequence identity with their C. crescentus homologues, while other homologue pairs tended to have lower levels of identity. In addition, the K31 chromosome contains more than 1600 genes with no homologue in NA1000.