Correction: Ribovirus classification by a polymerase barcode sequence.

[This corrects the article DOI: 10.7717/peerj.14055.].

Viroid-like colonists of human microbiomes.

Here, we describe the "Obelisks," a previously unrecognised class of viroid-like elements that we first identified in human gut metatranscriptomic data. "Obelisks" share several properties: (i) apparently circular RNA ~1kb genome assemblies, (ii) predicted rod-like secondary structures encompassing the entire genome, and (iii) open reading frames coding for a novel protein superfamily, which we call the "Oblins". We find that Obelisks form their own distinct phylogenetic group with no detectable sequence or structural similarity to known biological agents. Further, Obelisks are prevalent in tested human microbiome metatranscriptomes with representatives detected in ~7% of analysed stool metatranscriptomes (29/440) and in ~50% of analysed oral metatranscriptomes (17/32). Obelisk compositions appear to differ between the anatomic sites and are capable of persisting in individuals, with continued presence over >300 days observed in one case. Large scale searches identified 29,959 Obelisks (clustered at 90% nucleotide identity), with examples from all seven continents and in diverse ecological niches. From this search, a subset of Obelisks are identified to code for Obelisk-specific variants of the hammerhead type-III self-cleaving ribozyme. Lastly, we identified one case of a bacterial species (Streptococcus sanguinis) in which a subset of defined laboratory strains harboured a specific Obelisk RNA population. As such, Obelisks comprise a class of diverse RNAs that have colonised, and gone unnoticed in, human, and global microbiomes.

Known phyla dominate the Tara Oceans RNA virome.

A recent study proposed five new RNA virus phyla, two of which, 'Taraviricota' and 'Arctiviricota', were stated to be 'dominant in the oceans'. However, the study's assignments classify 28,353 putative RdRp-containing contigs to known phyla but only 886 (2.8%) to the five proposed new phyla combined. I re-mapped the reads to the contigs, finding that known phyla also account for a large majority (93.8%) of reads according to the study's classifications, and that contigs originally assigned to 'Arctiviricota' accounted for only a tiny fraction (0.01%) of reads from Arctic Ocean samples. Performing my own virus identification and classifications, I found that 99.95 per cent of reads could be assigned to known phyla. The most abundant species was Beihai picorna-like virus 34 (15% of reads), and the most abundant order-like cluster was classified as Picornavirales (45% of reads). Sequences in the claimed new phylum 'Pomiviricota' were placed inside a phylogenetic tree for established order Durnavirales with 100 per cent confidence. Moreover, two contigs assigned to the proposed phylum 'Taraviricota' were found to have high-identity alignments to dinoflagellate proteins, tentatively identifying this group of RdRp-like sequences as deriving from non-viral transcripts. Together, these results comprehensively contradict the claim that new phyla dominate the data.

Hybrids of RNA viruses and viroid-like elements replicate in fungi.

Earth's life may have originated as self-replicating RNA, and it has been argued that RNA viruses and viroid-like elements are remnants of such pre-cellular RNA world. RNA viruses are defined by linear RNA genomes encoding an RNA-dependent RNA polymerase (RdRp), whereas viroid-like elements consist of small, single-stranded, circular RNA genomes that, in some cases, encode paired self-cleaving ribozymes. Here we show that the number of candidate viroid-like elements occurring in geographically and ecologically diverse niches is much higher than previously thought. We report that, amongst these circular genomes, fungal ambiviruses are viroid-like elements that undergo rolling circle replication and encode their own viral RdRp. Thus, ambiviruses are distinct infectious RNAs showing hybrid features of viroid-like RNAs and viruses. We also detected similar circular RNAs, containing active ribozymes and encoding RdRps, related to mitochondrial-like fungal viruses, highlighting fungi as an evolutionary hub for RNA viruses and viroid-like elements. Our findings point to a deep co-evolutionary history between RNA viruses and subviral elements and offer new perspectives in the origin and evolution of primordial infectious agents, and RNA life.

Differentiating methicillin resistant and susceptible Staphylococcus aureus from ocular infections using photoacoustic labeling.

Introduction: Antibiotic resistance in bacterial species constitutes a growing problem in the clinical management of infections. Not only does it limit therapeutic options, but application of ineffective antibiotics allows resistant species to progress prior to prescribing more effective treatment to patients. Methicillin resistance in Staphylococcus aureus is a major problem in clinical infections as it is the most common hospital acquired infection. Methods: We developed a photoacoustic flow cytometer using engineered bacteriophage as probes for rapid determination of methicillin resistance in Staphylococcus aureus with thirteen clinical samples obtained from keratitis patients. This method irradiates cells under flow with 532 nm laser light and selectively generates acoustic waves in labeled bacterial cells, thus enabling detection and enumeration of them. Staphylococcus aureus isolates were classified from culture isolation as either methicillin resistant or susceptible using cefoxitin disk diffusion testing. The photoacoustic method enumerates bacterial cells before and after treatment with antibiotics. Decreasing counts of bacteria after treatment indicate susceptible strains. We quantified the bacterial cells in the treated and untreated samples. Results: Using k-means clustering on the data, we achieved 100% concordance with the classification of Staphylococcus aureus resistance using culture. Discussion: Photoacoustics can be used to differentiate methicillin resistant and susceptible strains of bacteria from ocular infections. This method may be generalized to other bacterial species using appropriate bacteriophages and testing for resistance using other antibiotics.

Photoacoustic Flow Cytometry Using Functionalized Microspheres for Selective Detection of Bacteria.

Photoacoustic flow cytometry is a method to detect rare analytes in fluids. We developed photoacoustic flow cytometry to detect pathological cells in body fluids, such as circulating tumor cells or bacteria in blood. In order to induce specific optical absorption in bacteria, we use modified bacteriophage that precisely target bacterial species or subspecies for rapid identification. In order to reduce detection variability and to halt the lytic lifescycle that results in lysis of the bacteria, we attached dyed latex microspheres to the tail fibers of bacteriophage that retained the bacterial recognition binding sites. We tested these microsphere complexes using Salmonella enterica (Salmonella) and Escherichia coli (E. coli) bacteria and found robust and specific detection of targeted bacteria. In our work we used LT2, a strain of Salmonella, against K12, a strain of E. coli. Using Det7, a bacteriophage that binds to LT2 and not to K12, we detected an average of 109.3±9.0 of LT2 versus 2.0±1.7 of K12 using red microspheres and 86.7±13.2 of LT2 versus 0.3±0.6 of K12 using blue microspheres. These results confirmed our ability to selectively detect bacterial species using photoacoustic flow cytometry.

Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny.

Multiple sequence alignments are widely used to infer evolutionary relationships, enabling inferences of structure, function, and phylogeny. Standard practice is to construct one alignment by some preferred method and use it in further analysis; however, undetected alignment bias can be problematic. I describe Muscle5, a novel algorithm which constructs an ensemble of high-accuracy alignment with diverse biases by perturbing a hidden Markov model and permuting its guide tree. Confidence in an inference is assessed as the fraction of the ensemble which supports it. Applied to phylogenetic tree estimation, I show that ensembles can confidently resolve topologies with low bootstrap according to standard methods, and conversely that some topologies with high bootstraps are incorrect. Applied to the phylogeny of RNA viruses, ensemble analysis shows that recently adopted taxonomic phyla are probably polyphyletic. Ensemble analysis can improve confidence assessment in any inference from an alignment.

Ribovirus classification by a polymerase barcode sequence.

RNA viruses encoding a polymerase gene (riboviruses) dominate the known eukaryotic virome. High-throughput sequencing is revealing a wealth of new riboviruses known only from sequence, precluding classification by traditional taxonomic methods. Sequence classification is often based on polymerase sequences, but standardised methods to support this approach are currently lacking. To address this need, we describe the polymerase palmprint, a segment of the palm sub-domain robustly delineated by well-conserved catalytic motifs. We present an algorithm, Palmscan, which identifies palmprints in nucleotide and amino acid sequences; PALMdb, a collection of palmprints derived from public sequence databases; and palmID, a public website implementing palmprint identification, search, and annotation. Together, these methods demonstrate a proof-of-concept workflow for high-throughput characterisation of RNA viruses, paving the path for the continued rapid growth in RNA virus discovery anticipated in the coming decade.

GMP Manufacturing and IND-Enabling Studies of a Recombinant Hyperimmune Globulin Targeting SARS-CoV-2.

Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus.

Petabase-scale sequence alignment catalyses viral discovery.

Public databases contain a planetary collection of nucleic acid sequences, but their systematic exploration has been inhibited by a lack of efficient methods for searching this corpus, which (at the time of writing) exceeds 20 petabases and is growing exponentially1. Here we developed a cloud computing infrastructure, Serratus, to enable ultra-high-throughput sequence alignment at the petabase scale. We searched 5.7 million biologically diverse samples (10.2 petabases) for the hallmark gene RNA-dependent RNA polymerase and identified well over 105 novel RNA viruses, thereby expanding the number of known species by roughly an order of magnitude. We characterized novel viruses related to coronaviruses, hepatitis delta virus and huge phages, respectively, and analysed their environmental reservoirs. To catalyse the ongoing revolution of viral discovery, we established a free and comprehensive database of these data and tools. Expanding the known sequence diversity of viruses can reveal the evolutionary origins of emerging pathogens and improve pathogen surveillance for the anticipation and mitigation of future pandemics.

Photoacoustic discrimination of antibiotic-resistant and sensitive Staphylococcus aureus isolates.

OBJECTIVES: Bacteremia is a serious and potentially lethal condition. Staphylococcus aureus is a leading cause of bacteremia and methicillin-resistant S. aureus (MRSA) accounts for more than a third of the cases. Compared to methicillin-sensitive S. aureus, MRSA is more than twice as likely to be fatal. Furthermore, subpopulations of seemingly isogenic bacteria may exhibit a range of susceptibilities, often called heterogenous resistance. These heterogeneous antibiotic-resistant infections are often misdiagnosed as hospital-acquired secondary infections because there are no clinically used tests that can differentiate between homogeneous and heterogeneous antibiotic resistance. We describe the development and proof of concept of rapid bacterial identification using photoacoustic flow cytometry and labeled bacteriophages with the characterization and differentiation of heterogeneous antibiotic-resistant bacterial infections. METHODS: In photoacoustic flow cytometry, pulsed laser light is delivered to a sample flowing past a focused transducer and particles that absorb laser light create an acoustic response. Optically labeled bacteriophage are added to a bacterial mixture that flows through the photoacoustic chamber. The presence of target bacteria is determined by bound labeled phage which are detected photoacoustically. Incubation of bacterial samples in the presence and absence of the antibiotic daptomycin creates a difference in bacterial cell numbers that is quantified using photoacoustic flow cytometry. RESULTS: Four clinical isolates were tested in the presence and absence of daptomycin. Photoacoustic events for each isolate were recorded and compared to growth curves. Samples treated with daptomycin fell into three categories: resistant, susceptible, and heterogeneous resistant. CONCLUSIONS: Here we show a method to determine the presence of bacteria as a marker for bloodstream infection level and antibiotic sensitivity in less than 4 hours. Additionally, these results show an ability to identify heterogeneous resistant strains that are often misidentified.

Perceptions of self-medication of the population, during the covid-19 pandemic in Peru / Percepciones de automedicación de la población durante la pandemia covid-19 en Perú

Background: Describe characteristics of self-medication of the population, in the context of the COVID 19 pandemic. Material and Methods: We carried out a cross-sectional survey with students in a major public Peruvian university. We measured perception of self-medication of the population, as well as characteristics, consequences, and associated factors. Results: A total of 274 students of Pharmacy and biochemistry filled in the questionnaire, 80.3% of which reported to know someone that self-medicated during the COVID-19 pandemic. 98.9 % of surveyed participants considered that the current pandemic increased self-medication practices in the population and that it occurred more frequently when people have symptoms associated with COVID-19 (65.4%). The medicines most frequently used were ivermectin (79.2%), azithromycin (71.2%) and hydroxychloroquine (38.7%). Conclusion: According to the surveyed participants, self-medication has increased in the general population during the COVID-19 pandemic. Strategies to prevent these practices are needed, as they could delay adequate medical care.

Introducción: Describir las características de la automedicación de la población, en el contexto de la pandemia de COVID 19. Material y Metodos: Realizamos una encuesta transversal con estudiantes de una importante universidad pública peruana. Se midió la percepción de la automedicación de la población, así como características, consecuencias y factores asociados. Resultados: Completaron el cuestionario un total de 274 estudiantes de Farmacia y Bioquímica, de los cuales el 80,3% refirió conocer a alguien que se automedicó durante la pandemia de COVID-19. El 98,9% de los encuestados consideró que la pandemia actual aumentó las prácticas de automedicación en la población y que se presentó con mayor frecuencia cuando las personas presentan síntomas asociados al COVID-19 (65,4%). Los medicamentos más utilizados fueron ivermectina (79,2%), azitromicina (71,2%) e hidroxicloroquina (38,7%). Conclusión: Según los participantes encuestados, la automedicación ha aumentado en la población general durante la pandemia de COVID-19. Se necesitan estrategias para prevenir estas prácticas, ya que podrían retrasar la atención médica adecuada.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

Capture and Isolation of Circulating Melanoma Cells Using Photoacoustic Flowmetry.

Early detection of cancer has been a goal of cancer research in general and melanoma research in particular (Birnbaum et al., Lancet Glob Health 6:e885-e893, 2018; Alendar et al., Bosnian J Basic Med Sci 9:77-80, 2009). Early detection of metastasis has been targeted as pivotal to increasing survival rates (Menezes et al., Adv Cancer Res 132:1-44, 2016). Melanoma, though curable in its early stages, has a dramatic decrease in survival rates once metastasis has occurred (Sharma et al., Biotechnol Adv 36:1063-1078, 2018). The transition to metastasis is not well understood and is an area of increasing interest. Metastasis is always premeditated by the shedding of circulating tumor cells (CTCs) from the primary tumor. The ability to isolate rare CTCs from the bloodstream has led to a host of new targets and therapies for cancer (Micalizzi et al., Genes Dev 31:1827-1840, 2017). Detection of CTCs also allows for disease progression to be tracked in real time while eliminating the need to wait for additional tumors to grow. Using a photoacoustic flowmeter, in which we induce ultrasonic responses from circulating melanoma cells (CMCs), we identify and quantify these cells in order to track disease progression. Additionally, these CMCs are captured and isolated allowing for future analysis such as RNA-Seq or microarray analysis.

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action.

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI-40 13-am43 and L cII-101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 72 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (immI: ant, arc) or known genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ∼35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).

Syncmers are more sensitive than minimizers for selecting conserved k­mers in biological sequences.

Minimizers are widely used to select subsets of fixed-length substrings (k-mers) from biological sequences in applications ranging from read mapping to taxonomy prediction and indexing of large datasets. The minimizer of a string of w consecutive k-mers is the k-mer with smallest value according to an ordering of all k-mers. Syncmers are defined here as a family of alternative methods which select k-mers by inspecting the position of the smallest-valued substring of length s < k within the k-mer. For example, a closed syncmer is selected if its smallest s-mer is at the start or end of the k-mer. At least one closed syncmer must be found in every window of length (k - s) k-mers. Unlike a minimizer, a syncmer is identified by its sequence alone, and is therefore synchronized in the following sense: if a given k-mer is selected from one sequence, it will also be selected from any other sequence. Also, minimizers can be deleted by mutations in flanking sequence, which cannot happen with syncmers. Experiments on minimizers with parameters used in the minimap2 read mapper and Kraken taxonomy prediction algorithm respectively show that syncmers can simultaneously achieve both lower density and higher conservation compared to minimizers.

Predicting Metastasis in Melanoma by Enumerating Circulating Tumor Cells Using Photoacoustic Flow Cytometry.

BACKGROUND AND OBJECTIVES: Enumerating circulating tumor cells has been used as a method of monitoring progression of various cancers. Various methods for detecting circulating melanoma cells (CMCs) have been reported, but none has had sufficient sensitivity to determine if the presence of rare CMCs in the blood of Stage I-III melanoma patients predicts if those patients eventually develop metastatic disease. STUDY DESIGN: We quantified CMCs in serial blood samples from 38 early stage melanoma patients to determine if CMC numbers predict development of metastatic melanoma. CMCs were enumerated using a photoacoustic flow cytometric detection system that uses a laser to induce high frequency acoustic signals in pigmented CMCs. RESULTS: We observed that detection of greater than 2 CMCs/ml of blood from patients with Stage I-III melanoma predicts metastatic disease. Of the 11 patients we studied who had two or fewer CMCs detected at all time points tested, none progressed to metastatic disease over a mean follow-up of 1288 days. In contrast, 18 of the 27 patients (67%) having more than 2 CMCs/ml at one or more time points progressed to metastatic disease over a mean follow-up of 850 days. CONCLUSIONS: Photoacoustic flow cytometry can detect rare CMCs in the blood of Stage I-III melanoma patients and detectionof these cells is predictive of subsequent development of metastatic disease. Lasers Surg. Med.

URMAP, an ultra-fast read mapper.

Mapping of reads to reference sequences is an essential step in a wide range of biological studies. The large size of datasets generated with next-generation sequencing technologies motivates the development of fast mapping software. Here, I describe URMAP, a new read mapping algorithm. URMAP is an order of magnitude faster than BWA with comparable accuracy on several validation tests. On a Genome in a Bottle (GIAB) variant calling test with 30× coverage 2×150 reads, URMAP achieves high accuracy (precision 0.998, sensitivity 0.982 and F-measure 0.990) with the strelka2 caller. However, GIAB reference variants are shown to be biased against repetitive regions which are difficult to map and may therefore pose an unrealistically easy challenge to read mappers and variant callers.

Massively parallel interrogation and mining of natively paired human TCRαß repertoires.

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαß clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαß-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαß clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

Photoacoustic detection of circulating melanoma cells in late stage patients.

Melanoma is the deadliest skin cancer and is responsible for over 7000 deaths in the US annually. The spread of cancer, or metastasis, is responsible for these deaths, as secondary tumors interrupt normal organ function. Circulating tumor cells, or those cells that spread throughout the body from the primary tumor, are thought to be responsible for metastasis. We developed an optical method, photoacoustic flow cytometry, in order to detect and enumerate circulating melanoma cells (CMCs) from blood samples of patients. We tested the blood of Stage IV melanoma patients to show the ability of the photoacoustic flow cytometer to detect these rare cells in blood. We then tested the system on archived blood samples from Stage III melanoma patients with known outcomes to determine if detection of CMCs can predict future metastasis. We detected between 0 and 66 CMCs in Stage IV patients. For the Stage III study, we found that of those samples with CMCs, 2 remained disease free and 5 developed metastasis. Of those without CMCs, 6 remained disease free and 1 developed metastasis. We believe that photoacoustic detection of CMCs provides valuable information for the prediction of metastasis and we postulate a system for more accurate prognosis.