The reversible activation of norovirus by metal ions.

Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.

Molecular Surveillance for Lymphoproliferative Disease Virus and Reticuloendotheliosis Virus in Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) in Texas, USA.

Reticuloendotheliosis virus (REV) and lymphoproliferative disease virus (LPDV) are avian retroviruses that can cause neoplastic disease and present with similar pathologies. Lymphoproliferative disease virus has been reported in the Eastern US and states bordering Texas, USA, but has not been previously detected within the state. In a prior study, we detected REV in native Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) and an Eastern Wild Turkey (Meleagris gallopavo silvestris) originating from West Virginia. Given LPDV detection in states bordering Texas and our finding of an REV-positive Eastern Wild Turkey imported from a LPDV endemic region, we sought to determine LPDV prevalence in Texas and continue surveillance for REV. During 2018-20, dried blood spots from 373 individual Rio Grande Wild Turkeys from 20 different counties were tested for the presence of proviral REV or LPDV DNA. In affected counties, approximately 4% of individuals were infected with REV (7/197) or LPDV (10/273) and one bird was coinfected with both viruses. Phylogenetic analysis indicated a close relationship of the LPDV isolates to variants from other Southern and Central states. This study provides molecular evidence of LPDV in Texas, and continued surveillance is necessary to determine the potential effects of the virus on reproductive success, coinfections, and overall health of Wild Turkey populations.

Genome Sequence of Fowlpox Virus-Integrated Reticuloendotheliosis Virus from a Rio Grande Wild Turkey (Meleagris gallopavo intermedia).

We report the genome sequence of a nearly intact reticuloendotheliosis virus (REV) insertion within a field strain of fowlpox virus from a Rio Grande wild turkey in Gillespie County, TX. The proviral REV genome comprises 7,943 bp and contains partial long terminal repeats.

Near-Complete Proviral Genome Sequence of Reticuloendotheliosis Virus Isolated from an Attwater's Prairie Chicken (Tympanuchus cupido attwateri).

We report the near-complete proviral genome sequence of a reticuloendotheliosis virus isolated and propagated from an endangered Attwater's prairie chicken (Tympanuchus cupido attwateri) during a 2016-2017 outbreak at a captive breeding facility.

Complete Genome Sequence of Bacteriophage Fizzles, Isolated from Microbacterium foliorum.

Microbacteriophage Fizzles has a 62,078-bp linear double-stranded DNA genome sequence, predicted to contain 104 protein-coding genes. Fizzles is a Siphoviridae actinobacteriophage isolated from an ant hill soil sample collected in Stephenville, TX. Microbacteriophage Fizzles has >83.6% nucleotide identity with microbacteriophages Squash and Nike.

Complete Genome Sequence of Bacteriophage IndyLu, Isolated from a Microbacterium foliorum Culture.

Microbacteriophage IndyLu was isolated from Microbacterium foliorum NRRL B-24224. The 41,958-bp double-stranded DNA genome has 71 predicted protein coding genes and 1 tRNA. The lytic actinobacteriophage was extracted from soil samples collected in Stephenville, TX, and is related to cluster EB bacteriophages Didgeridoo and Lahqtemish.

Complete Genome Sequences of Mycobacterium Phages Tripl3t and Zeuska.

Tripl3t and Zeuska are siphoviral bacteriophages that were isolated from Mycobacterium smegmatis mc2 155 and contain double-stranded DNA genomes 53,565 bp and 53,598 bp in length, respectively. Tripl3t and Zeuska were annotated by students at Bluff Dale High School (Bluff Dale, TX) and Tolar High School (Tolar, TX) in community engagement with Tarleton State University.

Complete Genome Sequence of Mycobacteriophage Joy99.

Joy99 is a siphoviral mycobacteriophage with a 59,837-base pair double-stranded DNA genome and is predicted to contain 97 protein-coding genes and a single tRNA gene. Joy99 was isolated in Saint Louis, MO, and annotated by students at Bluff Dale High School in community engagement with Tarleton State University.

Complete Genome Sequence of Bacteriophage Finny, Isolated from a Microbacterium foliorum Culture.

Actinobacteriophage Finny contains a circularly permuted 40,313-bp double-stranded DNA genome with 63 predicted protein-coding genes. Finny was directly isolated from a soil sample collected in New Braunfels, Texas, that was incubated with Microbacterium foliorum SEA B-24224. Finny is closely related to bacteriophages MCubed, Andromedas, ColaCorta, Eleri, and Sansa.

Survey of Reticuloendotheliosis Virus in Wild Turkeys (Meleagris gallopavo) in Texas, USA.

Reticuloendotheliosis virus (REV) is an immunosuppressive and sometimes oncogenic avian retrovirus that establishes lifelong infection in a wide range of avian species. REV-infected wild birds roaming near at-risk captive flocks, such as is the case for the highly endangered Attwater's Prairie Chicken (APC; Tympanuchus cupido attwateri), could act as a reservoir for viral transmission. In wild birds, prevalence rates of REV are low and appearance of associated disease is uncommon. During 2016-17, nearly half of all captive adult APC mortality at Fossil Rim Wildlife Center captive breeding facility in Glen Rose, Texas, US was attributed to REV infection. The unusually high REV prevalence rate prompted us to survey for this virus in wild galliforms throughout the region. From 2016-17, 393 blood samples collected from two subspecies of Wild Turkeys (Meleagris gallopavo) were tested for REV proviral DNA through amplification of the viral 3' long terminal repeat and segments of the viral pol gene. In REV-affected counties, 5% (5/98) of native Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) were identified as REV-positive. In addition, we detected REV in one of 62 Eastern Wild Turkeys (Meleagris gallopavo silvestris) that had been imported during conservation efforts. To better determine protective measures, continued surveillance, including collection and genetic analysis of REV-infected samples, is necessary to identify sources of REV outbreaks in captive APC flocks.

The complete genome sequence of the methanogenic archaeon ISO4-H5 provides insights into the methylotrophic lifestyle of a ruminal representative of the Methanomassiliicoccales.

Methane emissions from agriculture represent around 9 % of global anthropogenic greenhouse emissions. The single largest source of this methane is animal enteric fermentation, predominantly from ruminant livestock where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens. In order to reduce methane emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop methane mitigation technologies. To gain insights into the role of methylotrophic methanogens in the rumen environment, the genome of a methanogenic archaeon has been sequenced. This isolate, strain ISO4-H5, was isolated from the ovine rumen and belongs to the order Methanomassiliicoccales. Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M, and so likely requires external coenzyme M to survive.

Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches.

Taxonomic characterization of active gastrointestinal microbiota is essential to detect shifts in microbial communities and functions under various conditions. This study aimed to identify and quantify potentially active rumen microbiota using total RNA sequencing and to compare the outcomes of this approach with the widely used targeted RNA/DNA amplicon sequencing technique. Total RNA isolated from rumen digesta samples from five beef steers was subjected to Illumina paired-end sequencing (RNA-seq), and bacterial and archaeal amplicons of partial 16S rRNA/rDNA were subjected to 454 pyrosequencing (RNA/DNA Amplicon-seq). Taxonomic assessments of the RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq datasets were performed using a pipeline developed in house. The detected major microbial phylotypes were common among the three datasets, with seven bacterial phyla, fifteen bacterial families, and five archaeal taxa commonly identified across all datasets. There were also unique microbial taxa detected in each dataset. Elusimicrobia and Verrucomicrobia phyla; Desulfovibrionaceae, Elusimicrobiaceae, and Sphaerochaetaceae families; and Methanobrevibacter woesei were only detected in the RNA-Seq and RNA Amplicon-seq datasets, whereas Streptococcaceae was only detected in the DNA Amplicon-seq dataset. In addition, the relative abundances of four bacterial phyla, eight bacterial families and one archaeal taxon were different among the three datasets. This is the first study to compare the outcomes of rumen microbiota profiling between RNA-seq and RNA/DNA Amplicon-seq datasets. Our results illustrate the differences between these methods in characterizing microbiota both qualitatively and quantitatively for the same sample, and so caution must be exercised when comparing data.

Complete Genome Sequence of Methanogenic Archaeon ISO4-G1, a Member of the Methanomassiliicoccales, Isolated from a Sheep Rumen.

Methanogenic archaeon ISO4-G1 is a methylotrophic methanogen belonging to the orderMethanomassiliicoccalesthat was isolated from a sheep rumen. Its genome has been sequenced to provide information on the genetic diversity of rumen methanogens in order to develop technologies for ruminant methane mitigation.

Draft Genome Sequence of the Rumen Methanogen Methanobrevibacter olleyae YLM1.

Methanobrevibacter olleyaeYLM1 is a hydrogenotrophic methanogen, isolated from the rumen of a lamb. Its genome has been sequenced to provide information on the genomic diversity of rumen methanogens and support the development of approaches to reduce methane formation by ruminants.

Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range.

Ruminant livestock are important sources of human food and global greenhouse gas emissions. Feed degradation and methane formation by ruminants rely on metabolic interactions between rumen microbes and affect ruminant productivity. Rumen and camelid foregut microbial community composition was determined in 742 samples from 32 animal species and 35 countries, to estimate if this was influenced by diet, host species, or geography. Similar bacteria and archaea dominated in nearly all samples, while protozoal communities were more variable. The dominant bacteria are poorly characterised, but the methanogenic archaea are better known and highly conserved across the world. This universality and limited diversity could make it possible to mitigate methane emissions by developing strategies that target the few dominant methanogens. Differences in microbial community compositions were predominantly attributable to diet, with the host being less influential. There were few strong co-occurrence patterns between microbes, suggesting that major metabolic interactions are non-selective rather than specific.

Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions.

In this study, methanogen-specific coenzyme F420 autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis.

Lambs fed fresh winter forage rape (Brassica napus L.) emit less methane than those fed perennial ryegrass (Lolium perenne L.), and possible mechanisms behind the difference.

The objectives of this study were to examine long-term effects of feeding forage rape (Brassica napus L.) on methane yields (g methane per kg of feed dry matter intake), and to propose mechanisms that may be responsible for lower emissions from lambs fed forage rape compared to perennial ryegrass (Lolium perenne L.). The lambs were fed fresh winter forage rape or ryegrass as their sole diet for 15 weeks. Methane yields were measured using open circuit respiration chambers, and were 22-30% smaller from forage rape than from ryegrass (averages of 13.6 g versus 19.5 g after 7 weeks, and 17.8 g versus 22.9 g after 15 weeks). The difference therefore persisted consistently for at least 3 months. The smaller methane yields from forage rape were not related to nitrate or sulfate in the feed, which might act as alternative electron acceptors, or to the levels of the potential inhibitors glucosinolates and S-methyl L-cysteine sulfoxide. Ruminal microbial communities in forage rape-fed lambs were different from those in ryegrass-fed lambs, with greater proportions of potentially propionate-forming bacteria, and were consistent with less hydrogen and hence less methane being produced during fermentation. The molar proportions of ruminal acetate were smaller and those of propionate were greater in forage rape-fed lambs, consistent with the larger propionate-forming populations and less hydrogen production. Forage rape contained more readily fermentable carbohydrates and less structural carbohydrates than ryegrass, and was more rapidly degraded in the rumen, which might favour this fermentation profile. The ruminal pH was lower in forage rape-fed lambs, which might inhibit methanogenic activity, shifting the rumen fermentation to more propionate and less hydrogen and methane. The significance of these two mechanisms remains to be investigated. The results suggest that forage rape is a potential methane mitigation tool in pastoral-based sheep production systems.

Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.