Metagenomic characterization of swine slurry in a North American swine farm operation

Abstract Modern day large-scale, high-density farming environments are inherently susceptible to viral outbreaks, inadvertently creating conditions that favor increased pathogen transmission and potential zoonotic spread. Metagenomic sequencing has proven to be a useful tool for characterizing the microbial burden in both people, livestock, and environmental samples. International efforts have been successful at characterizing pathogens in commercial farming environments, especially swine farms, however it is unclear whether the full extent of microbial agents have been adequately captured or is representative of farms elsewhere. To augment international efforts we performed metagenomic next-generation sequencing on nine swine slurry and three environmental samples from a United States of America (U.S.A.) farm operation, characterized the microbial composition of slurry, and identified novel viruses. We assembled a remarkable total of 1792 viral genomes, of which 554 were novel/divergent. We assembled 1637 Picobirnavirus genome segments, of which 538 are novel. In addition, we discovered 10 new viruses belonging to a novel taxon: porcine Statoviruses; which have only been previously reported in human, macaques, mouse, and cows. We assembled 3 divergent Posaviruses and 3 swine Picornaviruses. In addition to viruses described, we found other eukaryotic genera such as Entamoeba and Blastocystis, and bacterial genera such as Listeria, Treponema, Peptoclostridium and Bordetella in the slurry. Of these, two species Entamoeba histolytica and Listeria monocytogenes known to cause human disease were detected. Further, antimicrobial resistance genes such as tetracycline and MLS (macrolide, lincosamide, streptogramin) were also identified. Metagenomic surveillance in swine fecal slurry has great potential for novel and antimicrobial resistant pathogen detection

Evidence for sodium-rich alkaline water in the Tagish Lake parent body and implications for amino acid synthesis and racemization

Understanding the timing and mechanisms of amino acid synthesis and racemization on asteroidal parent bodies is key to demonstrating how amino acids evolved to be mostly left-handed in living organisms on Earth. It has been postulated that racemization can occur rapidly dependent on several factors, including the pH of the aqueous solution. Here, we conduct nanoscale geochemical analysis of a framboidal magnetite grain within the Tagish Lake carbonaceous chondrite to demonstrate that the interlocking crystal arrangement formed within a sodium-rich, alkaline fluid environment. Notably, we report on the discovery of Na-enriched subgrain boundaries and nanometer-scale Ca and Mg layers surrounding individual framboids. These interstitial coatings would yield a surface charge state of zero in more-alkaline fluids and prevent assimilation of the individual framboids into a single grain. This basic solution would support rapid synthesis and racemization rates on the order of years, suggesting that the low abundances of amino acids in Tagish Lake cannot be ascribed to fluid chemistry

Conformational adaptation of Asian macaque TRIMCyp directs lineage specific antiviral activity

TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5α but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations

Biochemical Characterization of a Recombinant TRIM5 Protein That Restricts Human Immunodeficiency Virus Type 1 Replication

The rhesus monkey intrinsic immunity factor TRIM5αrh recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5α proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5αrh protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5α proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5α proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5α proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins

Genomic and serologic characterization of enterovirus A71 brainstem encephalitis

OBJECTIVE: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. METHODS: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. RESULTS: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. CONCLUSION: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.Grants supporting this project include the National Multiple Sclerosis Society and the American Academy of Neurology award FAN-1608-25607 (R.D.S.), Clinical Research Training Scholarship P0534134 (P.S.R.), Sandler and William K. Bowes Jr Foundations (M.R.W., J.L.D., L.M.K., H.A.S., K.C.Z.), Rachleff Family Foundation (M.R.W.), and NINDS of the NIH under award K08NS096117 (M.R.W.) and F31NS113432 (K.E.L.). This study was partially supported by a grant from the Spanish National Health Institute [grant number PI15CIII-00020] and the European Regional Development Fund (FEDER funds). UCSF Biomedical Sciences Program (I.A.H., K.E.L.), UCSF Medical Scientist Training Program (K.E.L.), and the Chan Zuckerberg Biohub (J.E.P., W.W., C.K.C., J.L.D., E.D.C.) also supported this project.S

Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children

This article is made available for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing.BACKGROUND: Despite improved diagnostics, pulmonary pathogens in immunocompromised children frequently evade detection, leading to significant mortality. Therefore, we aimed to develop a highly sensitive metagenomic next-generation sequencing (mNGS) assay capable of evaluating the pulmonary microbiome and identifying diverse pathogens in the lungs of immunocompromised children. METHODS: We collected 41 lower respiratory specimens from 34 immunocompromised children undergoing evaluation for pulmonary disease at 3 children's hospitals from 2014-2016. Samples underwent mechanical homogenization, parallel RNA/DNA extraction, and metagenomic sequencing. Sequencing reads were aligned to the National Center for Biotechnology Information nucleotide reference database to determine taxonomic identities. Statistical outliers were determined based on abundance within each sample and relative to other samples in the cohort. RESULTS: We identified a rich cross-domain pulmonary microbiome that contained bacteria, fungi, RNA viruses, and DNA viruses in each patient. Potentially pathogenic bacteria were ubiquitous among samples but could be distinguished as possible causes of disease by parsing for outlier organisms. Samples with bacterial outliers had significantly depressed alpha-diversity (median, 0.61; interquartile range [IQR], 0.33-0.72 vs median, 0.96; IQR, 0.94-0.96; P < .001). Potential pathogens were detected in half of samples previously negative by clinical diagnostics, demonstrating increased sensitivity for missed pulmonary pathogens (P < .001). CONCLUSIONS: An optimized mNGS assay for pulmonary microbes demonstrates significant inoculation of the lower airways of immunocompromised children with diverse bacteria, fungi, and viruses. Potential pathogens can be identified based on absolute and relative abundance. Ongoing investigation is needed to determine the pathogenic significance of outlier microbes in the lungs of immunocompromised children with pulmonary disease

Barrier-to-autointegration factor 1 (Banf1) regulates poly [ADP-ribose] polymerase 1 (PARP1) activity following oxidative DNA damage

The DNA repair capacity of human cells declines with age, in a process that is not clearly understood. Mutation of the nuclear envelope protein barrier-to-autointegration factor 1 (Banf1) has previously been shown to cause a human progeroid disorder, Néstor–Guillermo progeria syndrome (NGPS). The underlying links between Banf1, DNA repair and the ageing process are unknown. Here, we report that Banf1 controls the DNA damage response to oxidative stress via regulation of poly [ADP-ribose] polymerase 1 (PARP1). Specifically, oxidative lesions promote direct binding of Banf1 to PARP1, a critical NAD-dependent DNA repair protein, leading to inhibition of PARP1 auto-ADP-ribosylation and defective repair of oxidative lesions, in cells with increased Banf1. Consistent with this, cells from patients with NGPS have defective PARP1 activity and impaired repair of oxidative lesions. These data support a model whereby Banf1 is crucial to reset oxidative-stress-induced PARP1 activity. Together, these data offer insight into Banf1-regulated, PARP1-directed repair of oxidative lesions

TRIM5 Suppresses Cross-Species Transmission of a Primate Immunodeficiency Virus and Selects for Emergence of Resistant Variants in the New Species

Cross-species transmission of simian immunodeficiency virus from sooty mangabeys (SIVsm) into rhesus macaques, and subsequent emergence of pathogenic SIVmac, required adaptation to overcome restriction encoded by the macaque TRIM5 gene

Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients:a feasibility and clinical validity study

BACKGROUND: Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens. METHODS: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. RESULTS: Human DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. CONCLUSIONS: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures, and clinically actionable information obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes

Antibiotics Threaten Wildlife: Circulating Quinolone Residues and Disease in Avian Scavengers

Antibiotic residues that may be present in carcasses of medicated livestock could pass to and greatly reduce scavenger wildlife populations. We surveyed residues of the quinolones enrofloxacin and its metabolite ciprofloxacin and other antibiotics (amoxicillin and oxytetracycline) in nestling griffon Gyps fulvus, cinereous Aegypius monachus and Egyptian Neophron percnopterus vultures in central Spain. We found high concentrations of antibiotics in the plasma of many nestling cinereous (57%) and Egyptian (40%) vultures. Enrofloxacin and ciprofloxacin were also found in liver samples of all dead cinereous vultures. This is the first report of antibiotic residues in wildlife. We also provide evidence of a direct association between antibiotic residues, primarily quinolones, and severe disease due to bacterial and fungal pathogens. Our results indicate that, by damaging the liver and kidney and through the acquisition and proliferation of pathogens associated with the depletion of lymphoid organs, continuous exposure to antibiotics could increase mortality rates, at least in cinereous vultures. If antibiotics ingested with livestock carrion are clearly implicated in the decline of the vultures in central Spain then it should be considered a primary concern for conservation of their populations