Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

Single-Cell Approach Reveals Intercellular Heterogeneity in Phage-Producing Capacities.

Bacteriophage burst size is the average number of phage virions released from infected bacterial cells, and its magnitude depends on the duration of an intracellular progeny accumulation phase. Burst size is often measured at the population level, not the single-cell level, and consequently, statistical moments are not commonly available. In this study, we estimated the bacteriophage lambda (λ) single-cell burst size mean and variance following different intracellular accumulation period durations by employing Escherichia coli lysogens bearing lysis-deficient λ prophages. Single lysogens can be isolated and chemically lysed at desired times following prophage induction to quantify progeny intracellular accumulation within individual cells. Our data showed that λ phage burst size initially increased exponentially with increased lysis time (i.e., period between induction and chemical lysis) and then saturated at longer lysis times. We also demonstrated that cell-to-cell variation, or "noise," in lysis timing did not contribute significantly to burst size noise. The burst size noise remained constant with increasing mean burst size. The most likely explanation for the experimentally observed constant burst size noise was that cell-to-cell differences in burst size originated from intercellular heterogeneity in cellular capacities to produce phages. The mean burst size measured at different lysis times was positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicated that there are other factors in addition to cell size that determine this cellular capacity. IMPORTANCE Phages produce offspring by hijacking a cell's replicative machinery. Previously, it was noted that the variation in the number of phages produced by single infected cells far exceeded cell size variation. It was hypothesized that this variation is a consequence of variation in the timing of host cell lysis. Here, we show that cell-to-cell variation in lysis timing does not significantly contribute to the burst size variation. We suggest that the constant burst size variation across different host lysis times results from cell-to-cell differences in capacity to produce phages. We found that the mean burst size measured at different lysis times was positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicated that there are other factors in addition to cell size that determine this cellular capacity.

Rethinking the evolution of single-stranded RNA (ssRNA) bacteriophages based on genomic sequences and characterizations of two R-plasmid-dependent ssRNA phages, C-1 and Hgal1.

We have sequenced and characterized two R-plasmid-dependent single-stranded RNA bacteriophages (RPD ssRNA phages), C-1 and Hagl1. Phage C-1 requires a conjugative plasmid of the IncC group, while Hgal1 requires the IncH group. Both the adsorption rate constants and one-step growth curves are determined for both phages. We also empirically confirmed the lysis function of the predicted lysis genes. Genomic sequencing and phylogenetic analyses showed that both phages belong to the Levivirus group and are most closely related to another IncP-plasmid-dependent ssRNA phage, PRR1. Furthermore, our result strongly suggests that the stereotypical bauplans of genome organization found in Levivirus and Allolevivirus predate phage specialization for conjugative plasmids, suggesting that the utilization of conjugative plasmids for cell attachment and entry comprises independent evolutionary events for these two main clades of ssRNA phages. Our result is also consistent with findings of a previous study, making the Levivirus-like genome organization ancestral and the Allolevivirus-like genome derived. To obtain a deeper insight into the evolution of ssRNA phages, more phages specializing for various conjugative plasmids and infecting different bacterial species would be needed.

An Optimal Lysis Time Maximizes Bacteriophage Fitness in Quasi-Continuous Culture.

Optimality models have a checkered history in evolutionary biology. While optimality models have been successful in providing valuable insight into the evolution of a wide variety of biological traits, a common objection is that optimality models are overly simplistic and ignore organismal genetics. We revisit evolutionary optimization in the context of a major bacteriophage life history trait, lysis time. Lysis time refers to the period spanning phage infection of a host cell and its lysis, whereupon phage progenies are released. Lysis time, therefore, directly determines phage fecundity assuming progeny assembly does not exhaust host resources prior to lysis. Noting that previous tests of lysis time optimality rely on batch culture, we implemented a quasi-continuous culture system to observe productivity of a panel of isogenic phage λ genotypes differing in lysis time. We report that under our experimental conditions, λ phage productivity is maximized around optimal lysis times ranging from 60 to 100 min, and λ wildtype strain falls within this range. It would appear that natural selection on phage λ lysis time uncovered a set of genetic solutions that optimized progeny production in its ecological milieu relative to alternative genotypes. We discuss this finding in light of recent results that lysis time variation is also minimized in the strains with lysis times closer to the λ wild-type strain. IMPORTANCE Optimality theory presents the idea that natural selection acts on organismal traits to produce genotypes that maximize organismal survival and reproduction. As such, optimality theory is a valuable tool in guiding our understanding of the genetic constraints and tradeoffs organisms experience as their genotypes are selected to produce optimal solutions to biological problems. However, optimality theory is often critiqued as being overly simplistic and ignoring the roles of chance and history in the evolution of organismal traits. We show here that the wild-type genotype of a popular laboratory model organism, the bacteriophage λ, produces a phenotype for a major life history trait, lysis time, that maximizes the reproductive success of bearers of that genotype relative to other possible genotypes. This result demonstrates, as is rarely shown experimentally, that natural selection can achieve optimal solutions to ecological challenges.

Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater.

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID's EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitution. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from immunocompromised patients or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population. Author Summary: During the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source.

Effects of bacteriophage traits on plaque formation.

BACKGROUND: The appearance of plaques on a bacterial lawn is one of the enduring imageries in modern day biology. The seeming simplicity of a plaque has invited many hypotheses and models in trying to describe and explain the details of its formation. However, until now, there has been no systematic experimental exploration on how different bacteriophage (phage) traits may influence the formation of a plaque. In this study, we constructed a series of isogenic λ phages that differ in their adsorption rate, lysis timing, or morphology so that we can determine the effects if these changes on three plaque properties: size, progeny productivity, and phage concentration within plaques. RESULTS: We found that the adsorption rate has a diminishing, but negative impact on all three plaque measurements. Interestingly, there exists a concave relationship between the lysis time and plaque size, resulting in an apparent optimal lysis time that maximizes the plaque size. Although suggestive in appearance, we did not detect a significant effect of lysis time on plaque productivity. Nonetheless, the combined effects of plaque size and productivity resulted in an apparent convex relationship between the lysis time and phage concentration within plaques. Lastly, we found that virion morphology also affected plaque size. We compared our results to the available models on plaque size and productivity. For the models in their current forms, a few of them can capture the qualitative aspects of our results, but not consistently in both plaque properties. CONCLUSIONS: By using a collection of isogenic phage strains, we were able to investigate the effects of individual phage traits on plaque size, plaque productivity, and average phage concentration in a plaque while holding all other traits constant. The controlled nature of our study allowed us to test several model predictions on plaque size and plaque productivity. It seems that a more realistic theoretical approach to plaque formation is needed in order to capture the complex interaction between phage and its bacterium host in a spatially restricted environment.

Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater.

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.

Optimum Threshold Minimizes Noise in Timing of Intracellular Events.

How the noisy expression of regulatory proteins affects timing of intracellular events is an intriguing fundamental problem that influences diverse cellular processes. Here we use the bacteriophage λ to study event timing in individual cells where cell lysis is the result of expression and accumulation of a single protein (holin) in the Escherichia coli cell membrane up to a critical threshold level. Site-directed mutagenesis of the holin gene generated phage variants that vary in their lysis times from 30 to 190 min. Observation of the lysis times of single cells reveals an intriguing finding-the noise in lysis timing first decreases with increasing lysis time to reach a minimum and then sharply increases at longer lysis times. A mathematical model with stochastic expression of holin together with dilution from cell growth was sufficient to explain the non-monotonic noise profile and identify holin accumulation thresholds that generate precision in lysis timing.