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.

Are COVID-19 Vaccine Boosters Needed? The Science behind Boosters.

Waning vaccine-induced immunity coupled with the emergence of SARS-CoV-2 variants has led to increases in breakthrough infections, prompting consideration for vaccine booster doses. Boosters have been reported to be safe and increase SARS-CoV-2-specific neutralizing antibody levels, but how these doses impact the trajectory of the global pandemic and herd immunity is unknown. Information on immunology, epidemiology, and equitable vaccine distribution should be considered when deciding the timing and eligibility for COVID-19 vaccine boosters.

First-passage time approach to controlling noise in the timing of intracellular events.

In the noisy cellular environment, gene products are subject to inherent random fluctuations in copy numbers over time. How cells ensure precision in the timing of key intracellular events despite such stochasticity is an intriguing fundamental problem. We formulate event timing as a first-passage time problem, where an event is triggered when the level of a protein crosses a critical threshold for the first time. Analytical calculations are performed for the first-passage time distribution in stochastic models of gene expression. Derivation of these formulas motivates an interesting question: Is there an optimal feedback strategy to regulate the synthesis of a protein to ensure that an event will occur at a precise time, while minimizing deviations or noise about the mean? Counterintuitively, results show that for a stable long-lived protein, the optimal strategy is to express the protein at a constant rate without any feedback regulation, and any form of feedback (positive, negative, or any combination of them) will always amplify noise in event timing. In contrast, a positive feedback mechanism provides the highest precision in timing for an unstable protein. These theoretical results explain recent experimental observations of single-cell lysis times in bacteriophage [Formula: see text] Here, lysis of an infected bacterial cell is orchestrated by the expression and accumulation of a stable [Formula: see text] protein up to a threshold, and precision in timing is achieved via feedforward rather than feedback control. Our results have broad implications for diverse cellular processes that rely on precise temporal triggering of events.

What ecologists can tell virologists.

I pictured myself as a virus…and tried to sense what it would be like. --Jonas Salk. Ecology as a science evolved from natural history, the observational study of the interactions of plants and animals with each other and their environments. As natural history matured, it became increasingly quantitative, experimental, and taxonomically broad. Focus diversified beyond the Eukarya to include the hidden world of microbial life. Microbes, particularly viruses, were shown to exist in unfathomable numbers, affecting every living organism. Slowly viruses came to be viewed in an ecological context rather than as abstract, disease-causing agents. This shift is exemplified by an increasing tendency to refer to viruses as living organisms instead of inert particles. In recent years, researchers have recognized the critical contributions of viruses to fundamental ecological processes such as biogeochemical cycling, competition, community structuring, and horizontal gene transfer. This review describes virus ecology from a virus's perspective. If we are, like Jonas Salk, to imagine ourselves as a virus, what kind of world would we experience?

Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice.

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb-infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

Frequent coinfection reduces RNA virus population genetic diversity.

The masking of deleterious mutations by complementation and the reassortment of virus segments (virus sex) are expected to increase population genetic diversity among coinfecting viruses. Conversely, clonally reproducing or noncoinfecting virus populations may experience clonal interference where viral clones compete with one another, preventing selective sweeps. This dynamic reduces the efficiency of selection and increases the genetic diversity. To determine the relative influences of these forces on population genetic diversity, we evolved 6 populations of bacteriophage φ6 under conditions promoting or preventing coinfection. Following 300 generations, we isolated and partially sequenced 10 clones from each population. We found greater diversity among asexual populations than sexual populations. Moreover, sexual populations did not show greater relative fitnesses than asexual populations, implying that reduced genetic variation did not result from purifying selection. However, sexual populations were less genetically robust than asexual populations and likely more prone to the deleterious epistatic effects of mutations. As such, a neutral mutation on the asexually evolved (robust) background could be profoundly deleterious on the sexually evolved (brittle) background. This could facilitate sexual populations undergoing greater purifying selection to remove deleterious mutations, but this selection is not reflected by increases in average population fitness. Our results bolster a growing literature suggesting that RNA virus segmentation is probably not a mechanism that evolved because it provides a generalized benefit of sex.

When communication all changed.

Between early challenges and lasting opportunities, a deaf virologist reflects on how the pandemic transformed his access to academic spaces.

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.

Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice.

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we tested three bacteriophage strains for their Mtb-killing activities and found that two of them efficiently lysed Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently killed H37Rv in liquid culture and in Mtb-infected human primary macrophages. In subsequent experiments, we infected humanized mice with aerosolized H37Rv, then treated these mice with DS6A intravenously to test its in vivo efficacy. We found that DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduced Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrated the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

The prevalence of SARS-CoV-2 infection and other public health outcomes during the BA.2/BA.2.12.1 surge, New York City, April-May 2022.

BACKGROUND: Routine case surveillance data for SARS-CoV-2 are incomplete, unrepresentative, missing key variables of interest, and may be increasingly unreliable for timely surge detection and understanding the true burden of infection. METHODS: We conducted a cross-sectional survey of a representative sample of 1030 New York City (NYC) adult residents ≥18 years on May 7-8, 2022. We estimated the prevalence of SARS-CoV-2 infection during the preceding 14-day period. Respondents were asked about SARS-CoV-2 testing, testing outcomes, COVID-like symptoms, and contact with SARS-CoV-2 cases. SARS-CoV-2 prevalence estimates were age- and sex-adjusted to the 2020 U.S. POPULATION: We triangulated survey-based prevalence estimates with contemporaneous official SARS-CoV-2 counts of cases, hospitalizations, and deaths, as well as SARS-CoV-2 wastewater concentrations. RESULTS: We show that 22.1% (95% CI 17.9-26.2%) of respondents had SARS-CoV-2 infection during the two-week study period, corresponding to ~1.5 million adults (95% CI 1.3-1.8 million). The official SARS-CoV-2 case count during the study period is 51,218. Prevalence is estimated at 36.6% (95% CI 28.3-45.8%) among individuals with co-morbidities, 13.7% (95% CI 10.4-17.9%) among those 65+ years, and 15.3% (95% CI 9.6-23.5%) among unvaccinated persons. Among individuals with a SARS-CoV-2 infection, hybrid immunity (history of both vaccination and infection) is 66.2% (95% CI 55.7-76.7%), 44.1% (95% CI 33.0-55.1%) were aware of the antiviral nirmatrelvir/ritonavir, and 15.1% (95% CI 7.1-23.1%) reported receiving it. Hospitalizations, deaths and SARS-CoV-2 virus concentrations in wastewater remained well below that during the BA.1 surge. CONCLUSIONS: Our findings suggest that the true magnitude of NYC's BA.2/BA.2.12.1 surge may have been vastly underestimated by routine case counts and wastewater surveillance. Hybrid immunity, bolstered by the recent BA.1 surge, likely limited the severity of the BA.2/BA.2.12.1 surge.

It is difficult to assess the true prevalence of SARS-CoV-2, the virus that causes COVID-19, due to changes in testing practices and behaviors, including increasing at-home testing and decreasing healthcare provider-based testing. We conducted a population-representative survey in New York City to estimate the prevalence of SARS-CoV-2 during the second Omicron surge in spring 2022. We compared survey-based SARS-CoV-2 prevalence estimates with data on diagnosed cases, hospitalizations, deaths, and SARS-CoV-2 concentration in wastewater. Our survey-based estimates were nearly 30 times higher than official case counts and estimates of immunity among those with active infection were high. Taken together, our results suggest that the magnitude of the second Omicron surge was likely significantly underestimated, and high levels of immunity likely prevented a major surge in hospitalizations/deaths. Our findings might inform future work on COVID-19 surveillance and how to mitigate its spread.

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.

Humidity Reduces Rapid and Distant Airborne Dispersal of Viable Viral Particles in Classroom Settings.

The transmission of airborne pathogens is considered to be the main route through which a number of known and emerging respiratory diseases infect their hosts. While physical distancing and mask wearing may help mitigate short-range transmission, the extent of long-range transmission in closed spaces where a pathogen remains suspended in the air remains unknown. We have developed a method to detect viable virus particles by using an aerosolized bacteriophage Phi6 in combination with its host Pseudomonas phaseolicola, which when seeded on agar plates acts as a virus detector that can be placed at a range of distances away from an aerosol-generating source. By applying this method, we consistently detected viable phage particles at distances of up to 18 feet away from the source within 15 min of exposure in a classroom equipped with a state of the art HVAC system and determined that increasing the relative humidity beyond 40% significantly reduces dispersal. Our method, which can be further modified for use with other virus/host combinations, quantifies airborne transmission in the built environment and can thus be used to set safety standards for room capacity and to ascertain the efficacy of interventions in closed spaces of specified sizes and intended uses.

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.

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.

Differential bacteriophage mortality on exposure to copper.

Many studies report that copper can be used to control microbial growth, including that of viruses. We determined the rates of copper-mediated inactivation for a wide range of bacteriophages. We used two methods to test the effect of copper on bacteriophage survival. One method involved placing small volumes of bacteriophage lysate on copper and stainless steel coupons. Following exposure, metal coupons were rinsed with lysogeny broth, and the resulting fluid was serially diluted and plated on agar with the corresponding bacterial host. The second method involved adding copper sulfate (CuSO(4)) to bacteriophage lysates to a final concentration of 5 mM. Aliquots were removed from the mixture, serially diluted, and plated with the appropriate bacterial host. Significant mortality was observed among the double-stranded RNA (dsRNA) bacteriophages Φ6 and Φ8, the single-stranded RNA (ssRNA) bacteriophage PP7, the ssDNA bacteriophage ΦX174, and the dsDNA bacteriophage PM2. However, the dsDNA bacteriophages PRD1, T4, and λ were relatively unaffected by copper. Interestingly, lipid-containing bacteriophages were most susceptible to copper toxicity. In addition, in the first experimental method, the pattern of bacteriophage Φ6 survival over time showed a plateau in mortality after lysates dried out. This finding suggests that copper's effect on bacteriophage is mediated by the presence of water.

Factors influencing lysis time stochasticity in bacteriophage λ.

BACKGROUND: Despite identical genotypes and seemingly uniform environments, stochastic gene expression and other dynamic intracellular processes can produce considerable phenotypic diversity within clonal microbes. One trait that provides a good model to explore the molecular basis of stochastic variation is the timing of host lysis by bacteriophage (phage). RESULTS: Individual lysis events of thermally-inducible λ lysogens were observed using a temperature-controlled perfusion chamber mounted on an inverted microscope. Both mean lysis time (MLT) and its associated standard deviation (SD) were estimated. Using the SD as a measure of lysis time stochasticity, we showed that lysogenic cells in controlled environments varied widely in lysis times, and that the level of lysis time stochasticity depended on allelic variation in the holin sequence, late promoter (pR') activity, and host growth rate. In general, the MLT was positively correlated with the SD. Both lower pR' activities and lower host growth rates resulted in larger SDs. Results from premature lysis, induced by adding KCN at different time points after lysogen induction, showed a negative correlation between the timing of KCN addition and lysis time stochasticity. CONCLUSIONS: Taken together with results published by others, we conclude that a large fraction of λ lysis time stochasticity is the result of random events following the expression and diffusion of the holin protein. Consequently, factors influencing the timing of reaching critical holin concentrations in the cell membrane, such as holin production rate, strongly influence the mean lysis time and the lysis time stochasticity.

Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns.

Reassortment of the Rotavirus A (RVA) 11-segment dsRNA genome may generate new genome constellations that allow RVA to expand its host range or evade immune responses. Reassortment may also produce phylogenetic incongruities and weakly linked evolutionary histories across the 11 segments, obscuring reassortment-specific epistasis and changes in substitution rates. To determine the co-segregation patterns of RVA segments, we generated time-scaled phylogenetic trees for each of the 11 segments of 789 complete RVA genomes isolated from mammalian hosts and compared the segments' geodesic distances. We found that segments 4 (VP4) and 9 (VP7) occupied significantly different tree spaces from each other and from the rest of the genome. By contrast, segments 10 and 11 (NSP4 and NSP5/6) occupied nearly indistinguishable tree spaces, suggesting strong co-segregation. Host-species barriers appeared to vary by segment, with segment 9 (VP7) presenting the weakest association with host species. Bayesian Skyride plots were generated for each segment to compare relative genetic diversity among segments over time. All segments showed a dramatic decrease in diversity around 2007 coinciding with the introduction of RVA vaccines. To assess selection pressures, codon adaptation indices and relative codon deoptimization indices were calculated with respect to different host genomes. Codon usage varied by segment with segment 11 (NSP5) exhibiting significantly higher adaptation to host genomes. Furthermore, RVA codon usage patterns appeared optimized for expression in humans and birds relative to the other hosts examined, suggesting that translational efficiency is not a barrier in RVA zoonosis.

Why Do Antibiotics Exist?

In the struggle with antibiotic resistance, we are losing. There is now a serious threat of moving into a postantibiotic world. High levels of resistance, in terms of both frequency and strength, have evolved against all clinically approved antibiotics worldwide. The usable life span of new clinically approved antibiotics is typically less than a decade before resistance reaches frequencies so high as to require only guarded usage. However, microbes have produced antibiotics for millennia without resistance becoming an existential issue. If resistance is the inevitable consequence of antibiotic usage, as has been the human experience, why has it not become an issue for microbes as well, especially since resistance genes are as prevalent in nature as the genes responsible for antibiotic production? Here, we ask how antibiotics can exist given the almost ubiquitous presence of resistance genes in the very microbes that have produced and used antibiotics since before humans walked the planet. We find that the context of both production and usage of antibiotics by microbes may be key to understanding how resistance is managed over time, with antibiotic synthesis and resistance existing in a paired relationship, much like a cipher and key, that impacts microbial community assembly. Finally, we put forward the cohesive, ecologically based "secret society" hypothesis to explain the longevity of antibiotics in nature.