which permits unrestricted use, distribution, and reproduction in any medium, provided the origin... more which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The outcomes of host-parasite interactions depend on the coevolutionary forces acting upon them, but because every host-parasite relation is enmeshed in a web of biotic and abiotic interactions across a heterogeneous landscape, host-parasite coevolution has proven difficult to study. Simple laboratory phage-bacteria microcosms can ameliorate this difficulty by allowing controlled, well-replicated experiments with a limited number of interactors. Genetic, population, and life history data obtained from these studies permit a closer examination of the fundamental correlates of host-parasite coevolution. In this paper, I describe the results of phage-bacteria coevolutionary studies and their implications for the study of host-parasite coevolution. Recent experimental studies have confirmed phage-host coevolutionary dynamics in the laboratory and have shown that co...
These authors contributed equally to the work. Populations are at risk of extinction when unsuita... more These authors contributed equally to the work. Populations are at risk of extinction when unsuitable or when sink habitat exceeds a threshold frequency in the environment. Sinks that present cues associated with highquality habitats, termed ecological traps, have especially detrimental effects on net population growth at metapopulation scales. Ecological traps for viruses arise naturally, or can be engineered, via the expression of viral-binding sites on cells that preclude viral reproduction. We present a model for virus population growth in a heterogeneous host community, parameterized with data from populations of the RNA bacteriophage U6 presented with mixtures of suitable host bacteria and either neutral or trap cells. We demonstrate that viruses can sustain high rates of population growth in the presence of neutral non-hosts as long as some host cells are present, whereas trap cells dramatically reduce viral fitness. In addition, we demonstrate that the efficacy of traps for v...
Background: Despite identical genotypes and seemingly uniform environments, stochastic gene expre... more 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 l 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 ...
Waning vaccine-induced immunity coupled with the emergence of SARS-CoV-2 variants has led to incr... more 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.
Strains of Caenorhabditis elegans obtained from their natural soil environment exhibit one of two... more Strains of Caenorhabditis elegans obtained from their natural soil environment exhibit one of two forms of foraging behavior. Some strains forage solitarily and disperse evenly on a bacterial lawn. Other strains move rapidly until they encounter groups of conspecifics, and then slow their movement and join the group. Strains expressing these behaviors are globally widespread and have been isolated from the same location, suggesting a foraging polymorphism. We hypothesized that density-dependent selection maintains both foraging alleles in populations. Alternatively, both foraging alleles could be retained in populations through frequency-dependent selection. We tested both of these hypotheses by manipulating strain density and frequency, and observing changes in population density over time. Our results indicated that neither density- nor frequency-dependent selection appears to be responsible for the observed polymorphism. The clumping strain consistently out-competed the solitary ...
Background: Despite identical genotypes and seemingly uniform environments, stochastic gene expre... more 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 l 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 l...
Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may le... more 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 amplified regions of the SARS-CoV-2 Spike protein gene from RNA acquired from all 14 NYC wastewater treatment plants (WWTPs) and ascertained the diversity of lineages from these samples using high throughput sequencing. Here we report the detection and increasing frequencies of novel SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database. These lineages contain mutations rarely observed in clinical samples, including Q493K, Q498Y, H519N and T572N. Many of these mutations were found to expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. In addition, pseudoviruses containing the Spike amino acid sequence of these lineages were found to be r...
ABSTRACTThe following protocol describes our workflow for processing wastewater with the goal of ... more ABSTRACTThe 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. Furthermore, the RNA obtained using this protocol, minus the pasteurization step, can be sequenced both using a targeted approach sequencing specific regions or the whole genome. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to support their efforts in monitoring SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. Owing to a pasteurization step, it is safe for use in a BSL1+ facility. This step also increases the genet...
ABSTRACTMonitoring SARS-CoV-2 genetic diversity is strongly indicated because diversifying select... more ABSTRACTMonitoring 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 date, most data on SARS-CoV-2 genetic diversity has come from the sequencing of clinical samples, but such studies may suffer limitations due to costs and throughput. Wastewater-based epidemiology may provide an alternative and complementary approach for monitoring communities for novel variants. Given that SARS-CoV-2 can infect the cells of the human gut and is found in high concentrations in feces, wastewater may be a valuable source of SARS-CoV-2 RNA, which can be deep sequenced to provide information on the circulating variants in a community. Here we describe a safe, affordable protocol for the sequencing of SARS-CoV-2 RNA using high-throughput Illumina sequencing technology. Our targeted sequencing approach revealed the presence of mutations associated with several Varian...
Reassortment of the Rotavirus A (RVA) 11-segment dsRNA genome may generate new genome constellati... more 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 reassortant-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 treespaces from each other and from the rest of the genome. By contrast, segments 10 and 11 (NSP4 and NSP5/6) occupied nearly indistinguishable treespaces, suggesting strong co-segregation. Host-species barriers appeared to vary by segment, with segment 9 (VP7) presenting the least conservation by host species. Bayesia...
Because of their replication mode and segmented dsRNA genome, homologous recombination is assumed... more Because of their replication mode and segmented dsRNA genome, homologous recombination is assumed to be rare in the rotaviruses. We analyzed 23,627 complete rotavirus genome sequences available in the NCBI Virus Variation database and found numerous instances of homologous recombination, some of which prevailed across multiple sequenced isolates. In one case, recombination may have generated a novel rotavirus VP1 lineage. We also found strong evidence for intergenotypic recombination in which more than one sequence strongly supported the same event, particularly between different genotypes of the serotype protein, VP7. In addition, we found evidence for recombination in nine of eleven rotavirus A segments. Only two segments, 7 (NSP3) and 11 (NSP5/NSP6), did not show strongly supported evidence of prior recombination. The abundance of putative recombinants with substantial amino acid replacements occurring between different G types suggests a fitness advantage for the resulting recom...
Descriptions of resources, like the genome assemblies reported in Microbiology Resource Announcem... more Descriptions of resources, like the genome assemblies reported in Microbiology Resource Announcements, are often frozen at their time of publication, yet they will need to be interpreted in the midst of continually evolving technologies. It is therefore important to ensure that researchers accessing published resources have access to all of the information required to repeat, interpret, and extend these original analyses. Here, we provide a set of suggestions to help make certain that published resources remain useful and repeatable for the foreseeable future.
With estimated numbers greater than 10^31, viruses are the most abundant organisms on the planet,... more With estimated numbers greater than 10^31, viruses are the most abundant organisms on the planet, and occupy all habitats: aquatic, atmospheric and terrestrial. No cellular organisms - whether animal, plant or microbe - are free from viral parasitism. Consequently, the effects and influences of viruses are pervasive, directly or indirectly affecting all organisms, populations, communities and ecosystems. Here we consider the evolutionary ecology of the viruses of microorganisms (VoMs) which, due to the abundance of their hosts, outnumber all other types of viruses. Subfields of evolutionary ecology include life history evolution, population biology, biogeography, and community ecology. Like blind men describing an elephant, each approach only describes a facet of VoM evolutionary ecology. Here we describe some of the approaches used to describe VoM evolutionary ecology in hopes that a synthesis will allow some perception of the whole.
In an genomics course sponsored by the Howard Hughes Medical Institute (HHMI), undergraduate stud... more In an genomics course sponsored by the Howard Hughes Medical Institute (HHMI), undergraduate students have isolated and sequenced the genomes of more than 1,150 mycobacteriophages, creating the largest database of sequenced bacteriophages able to infect a single host, Mycobacterium smegmatis, a soil bacterium. Genomic analysis indicates that these mycobacteriophages can be grouped into 26 clusters based on genetic similarity. These clusters span a continuum of genetic diversity, with extensive genomic mosaicism among phages in different clusters. However, little is known regarding the primary hosts of these mycobacteriophages in their natural habitats, nor of their broader host ranges. As such, it is possible that the primary host of many newly isolated mycobacteriophages is not M. smegmatis, but instead a range of closely related bacterial species. However, determining mycobacteriophage host range presents difficulties associated with mycobacterial cultivability, pathogenicity and ...
which permits unrestricted use, distribution, and reproduction in any medium, provided the origin... more which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The outcomes of host-parasite interactions depend on the coevolutionary forces acting upon them, but because every host-parasite relation is enmeshed in a web of biotic and abiotic interactions across a heterogeneous landscape, host-parasite coevolution has proven difficult to study. Simple laboratory phage-bacteria microcosms can ameliorate this difficulty by allowing controlled, well-replicated experiments with a limited number of interactors. Genetic, population, and life history data obtained from these studies permit a closer examination of the fundamental correlates of host-parasite coevolution. In this paper, I describe the results of phage-bacteria coevolutionary studies and their implications for the study of host-parasite coevolution. Recent experimental studies have confirmed phage-host coevolutionary dynamics in the laboratory and have shown that co...
These authors contributed equally to the work. Populations are at risk of extinction when unsuita... more These authors contributed equally to the work. Populations are at risk of extinction when unsuitable or when sink habitat exceeds a threshold frequency in the environment. Sinks that present cues associated with highquality habitats, termed ecological traps, have especially detrimental effects on net population growth at metapopulation scales. Ecological traps for viruses arise naturally, or can be engineered, via the expression of viral-binding sites on cells that preclude viral reproduction. We present a model for virus population growth in a heterogeneous host community, parameterized with data from populations of the RNA bacteriophage U6 presented with mixtures of suitable host bacteria and either neutral or trap cells. We demonstrate that viruses can sustain high rates of population growth in the presence of neutral non-hosts as long as some host cells are present, whereas trap cells dramatically reduce viral fitness. In addition, we demonstrate that the efficacy of traps for v...
Background: Despite identical genotypes and seemingly uniform environments, stochastic gene expre... more 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 l 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 ...
Waning vaccine-induced immunity coupled with the emergence of SARS-CoV-2 variants has led to incr... more 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.
Strains of Caenorhabditis elegans obtained from their natural soil environment exhibit one of two... more Strains of Caenorhabditis elegans obtained from their natural soil environment exhibit one of two forms of foraging behavior. Some strains forage solitarily and disperse evenly on a bacterial lawn. Other strains move rapidly until they encounter groups of conspecifics, and then slow their movement and join the group. Strains expressing these behaviors are globally widespread and have been isolated from the same location, suggesting a foraging polymorphism. We hypothesized that density-dependent selection maintains both foraging alleles in populations. Alternatively, both foraging alleles could be retained in populations through frequency-dependent selection. We tested both of these hypotheses by manipulating strain density and frequency, and observing changes in population density over time. Our results indicated that neither density- nor frequency-dependent selection appears to be responsible for the observed polymorphism. The clumping strain consistently out-competed the solitary ...
Background: Despite identical genotypes and seemingly uniform environments, stochastic gene expre... more 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 l 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 l...
Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may le... more 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 amplified regions of the SARS-CoV-2 Spike protein gene from RNA acquired from all 14 NYC wastewater treatment plants (WWTPs) and ascertained the diversity of lineages from these samples using high throughput sequencing. Here we report the detection and increasing frequencies of novel SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database. These lineages contain mutations rarely observed in clinical samples, including Q493K, Q498Y, H519N and T572N. Many of these mutations were found to expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. In addition, pseudoviruses containing the Spike amino acid sequence of these lineages were found to be r...
ABSTRACTThe following protocol describes our workflow for processing wastewater with the goal of ... more ABSTRACTThe 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. Furthermore, the RNA obtained using this protocol, minus the pasteurization step, can be sequenced both using a targeted approach sequencing specific regions or the whole genome. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to support their efforts in monitoring SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. Owing to a pasteurization step, it is safe for use in a BSL1+ facility. This step also increases the genet...
ABSTRACTMonitoring SARS-CoV-2 genetic diversity is strongly indicated because diversifying select... more ABSTRACTMonitoring 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 date, most data on SARS-CoV-2 genetic diversity has come from the sequencing of clinical samples, but such studies may suffer limitations due to costs and throughput. Wastewater-based epidemiology may provide an alternative and complementary approach for monitoring communities for novel variants. Given that SARS-CoV-2 can infect the cells of the human gut and is found in high concentrations in feces, wastewater may be a valuable source of SARS-CoV-2 RNA, which can be deep sequenced to provide information on the circulating variants in a community. Here we describe a safe, affordable protocol for the sequencing of SARS-CoV-2 RNA using high-throughput Illumina sequencing technology. Our targeted sequencing approach revealed the presence of mutations associated with several Varian...
Reassortment of the Rotavirus A (RVA) 11-segment dsRNA genome may generate new genome constellati... more 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 reassortant-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 treespaces from each other and from the rest of the genome. By contrast, segments 10 and 11 (NSP4 and NSP5/6) occupied nearly indistinguishable treespaces, suggesting strong co-segregation. Host-species barriers appeared to vary by segment, with segment 9 (VP7) presenting the least conservation by host species. Bayesia...
Because of their replication mode and segmented dsRNA genome, homologous recombination is assumed... more Because of their replication mode and segmented dsRNA genome, homologous recombination is assumed to be rare in the rotaviruses. We analyzed 23,627 complete rotavirus genome sequences available in the NCBI Virus Variation database and found numerous instances of homologous recombination, some of which prevailed across multiple sequenced isolates. In one case, recombination may have generated a novel rotavirus VP1 lineage. We also found strong evidence for intergenotypic recombination in which more than one sequence strongly supported the same event, particularly between different genotypes of the serotype protein, VP7. In addition, we found evidence for recombination in nine of eleven rotavirus A segments. Only two segments, 7 (NSP3) and 11 (NSP5/NSP6), did not show strongly supported evidence of prior recombination. The abundance of putative recombinants with substantial amino acid replacements occurring between different G types suggests a fitness advantage for the resulting recom...
Descriptions of resources, like the genome assemblies reported in Microbiology Resource Announcem... more Descriptions of resources, like the genome assemblies reported in Microbiology Resource Announcements, are often frozen at their time of publication, yet they will need to be interpreted in the midst of continually evolving technologies. It is therefore important to ensure that researchers accessing published resources have access to all of the information required to repeat, interpret, and extend these original analyses. Here, we provide a set of suggestions to help make certain that published resources remain useful and repeatable for the foreseeable future.
With estimated numbers greater than 10^31, viruses are the most abundant organisms on the planet,... more With estimated numbers greater than 10^31, viruses are the most abundant organisms on the planet, and occupy all habitats: aquatic, atmospheric and terrestrial. No cellular organisms - whether animal, plant or microbe - are free from viral parasitism. Consequently, the effects and influences of viruses are pervasive, directly or indirectly affecting all organisms, populations, communities and ecosystems. Here we consider the evolutionary ecology of the viruses of microorganisms (VoMs) which, due to the abundance of their hosts, outnumber all other types of viruses. Subfields of evolutionary ecology include life history evolution, population biology, biogeography, and community ecology. Like blind men describing an elephant, each approach only describes a facet of VoM evolutionary ecology. Here we describe some of the approaches used to describe VoM evolutionary ecology in hopes that a synthesis will allow some perception of the whole.
In an genomics course sponsored by the Howard Hughes Medical Institute (HHMI), undergraduate stud... more In an genomics course sponsored by the Howard Hughes Medical Institute (HHMI), undergraduate students have isolated and sequenced the genomes of more than 1,150 mycobacteriophages, creating the largest database of sequenced bacteriophages able to infect a single host, Mycobacterium smegmatis, a soil bacterium. Genomic analysis indicates that these mycobacteriophages can be grouped into 26 clusters based on genetic similarity. These clusters span a continuum of genetic diversity, with extensive genomic mosaicism among phages in different clusters. However, little is known regarding the primary hosts of these mycobacteriophages in their natural habitats, nor of their broader host ranges. As such, it is possible that the primary host of many newly isolated mycobacteriophages is not M. smegmatis, but instead a range of closely related bacterial species. However, determining mycobacteriophage host range presents difficulties associated with mycobacterial cultivability, pathogenicity and ...
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