Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant.

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.

Clinical Metagenomic Sequencing for Diagnosis of Meningitis and Encephalitis.

BACKGROUND: Metagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test. METHODS: In a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review. RESULTS: We enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment. CONCLUSIONS: Routine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, NCT02910037.).

Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid.

Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for pan-pathogen detection, to be used clinically for diagnosis of neurological infections from CSF.

Introduction, Transmission Dynamics, and Fate of Early Severe Acute Respiratory Syndrome Coronavirus 2 Lineages in Santa Clara County, California.

We combined viral genome sequencing with contact tracing to investigate introduction and evolution of severe acute respiratory syndrome coronavirus 2 lineages in Santa Clara County, California, from 27 January to 21 March 2020. From 558 persons with coronavirus disease 2019, 101 genomes from 143 available clinical samples comprised 17 lineages, including SCC1 (n = 41), WA1 (n = 9; including the first 2 reported deaths in the United States, with postmortem diagnosis), D614G (n = 4), ancestral Wuhan Hu-1 (n = 21), and 13 others (n = 26). Public health intervention may have curtailed the persistence of lineages that appeared transiently during February and March. By August, only D614G lineages introduced after 21 March were circulating in Santa Clara County.

Associations of Early COVID-19 Cases in San Francisco With Domestic and International Travel.

In early-to-mid March 2020, 20 of 46 (43%) COVID-19 cases at a tertiary care hospital in San Francisco, California were travel related. Cases were significantly associated with travel to either Europe (odds ratio, 6.1) or New York (odds ratio, 32.9). Viral genomes recovered from 9 of 12 (75%) cases co-clustered with lineages circulating in Europe.

Metagenomic Next-Generation Sequencing of the 2014 Ebola Virus Disease Outbreak in the Democratic Republic of the Congo.

We applied metagenomic next-generation sequencing (mNGS) to detect Zaire Ebola virus (EBOV) and other potential pathogens from whole-blood samples from 70 patients with suspected Ebola hemorrhagic fever during a 2014 outbreak in Boende, Democratic Republic of the Congo (DRC) and correlated these findings with clinical symptoms. Twenty of 31 patients (64.5%) tested in Kinshasa, DRC, were EBOV positive by quantitative reverse transcriptase PCR (qRT-PCR). Despite partial degradation of sample RNA during shipping and handling, mNGS followed by EBOV-specific capture probe enrichment in a U.S. genomics laboratory identified EBOV reads in 22 of 70 samples (31.4%) versus in 21 of 70 (30.0%) EBOV-positive samples by repeat qRT-PCR (overall concordance = 87.1%). Reads from Plasmodium falciparum (malaria) were detected in 21 patients, of which at least 9 (42.9%) were coinfected with EBOV. Other positive viral detections included hepatitis B virus (n = 2), human pegivirus 1 (n = 2), Epstein-Barr virus (n = 9), and Orungo virus (n = 1), a virus in the Reoviridae family. The patient with Orungo virus infection presented with an acute febrile illness and died rapidly from massive hemorrhage and dehydration. Although the patient's blood sample was negative by EBOV qRT-PCR testing, identification of viral reads by mNGS confirmed the presence of EBOV coinfection. In total, 9 new EBOV genomes (3 complete genomes, and an additional 6 ≥50% complete) were assembled. Relaxed molecular clock phylogenetic analysis demonstrated a molecular evolutionary rate for the Boende strain 4 to 10× slower than that of other Ebola lineages. These results demonstrate the utility of mNGS in broad-based pathogen detection and outbreak surveillance.

Identification of high-risk human papillomavirus and Rb/E2F pathway genomic alterations in mutually exclusive subsets of colorectal neuroendocrine carcinoma.

Colorectal neuroendocrine carcinomas, both small cell and large cell types, are highly aggressive tumors with poor prognosis compared with colorectal adenocarcinoma. The molecular drivers of neuroendocrine carcinoma are best defined in small cell lung cancer, which shows near-universal genomic alterations in TP53 and RB1. The genetics of colorectal neuroendocrine carcinoma remain poorly understood; recent studies demonstrated infrequent RB1 alterations and genetics closely resembling colorectal adenocarcinoma. To better define the molecular pathogenesis of colorectal neuroendocrine carcinoma, we performed capture-based next-generation sequencing on 25 cases and evaluated for expression of p53, Rb, p16, and high-risk human papillomavirus (HR-HPV) subtypes using immunohistochemistry, in situ hybridization, and polymerase chain reaction. Rb/E2F pathway dysregulation was identified in nearly all cases (23/25, 92%) and occurred via three distinct mechanisms. First, RB1 genomic alteration was present in 56% (14/25) of cases and was accompanied by Rb protein loss, high p16 expression, and absence of HR-HPV; these cases also had frequent genomic alterations in TP53, the PI3K/Ras and Wnt pathways, as well as in DNA repair genes, with 4/14 cases being hypermutated. Second, 16% (4/25) of cases, all left-sided, had TP53 alteration without RB1 alteration; half of these harbored high-level amplifications in CCNE1 and MYC or MYCN and arose in patients with ulcerative colitis. Finally, 28% (7/25) of cases, all rectal or anal, lacked genomic alterations in RB1 or TP53 but were positive for HR-HPV. Our data demonstrate that Rb/E2F pathway dysregulation is essential in the pathogenesis of colorectal neuroendocrine carcinoma, akin to neuroendocrine carcinomas in other anatomic sites. Moreover, colorectal neuroendocrine carcinomas stratify into three distinct molecular subgroups, which can be differentiated based on Rb protein and HR-HPV status. HR-HPV infection represents a distinct mechanism for Rb and p53 inactivation in cases lacking genomic alterations in either gene. Differential treatment strategies for hypermutated and HPV-driven cases could improve patient outcomes.

Polyoma virus-associated carcinomas of the urologic tract: a clinicopathologic and molecular study.

In recent years, there has been increased interest in carcinomas of the urologic tract, that demonstrate association with the polyoma virus BK arising in immunosuppressed individuals, though the nature of this association is uncertain. To begin to understand this phenomenon, we reviewed the clinical, morphological, and immunohistochemical features of 11 carcinomas of the urologic tract, mainly urothelial (N = 9) and collecting duct carcinomas (N = 2), occurring during immunosuppression, and expressing polyoma virus T-antigen by immunohistochemistry. These were compared to a control group of carcinomas (N = 8), also arising during immunosuppression, but without T-antigen expression. A subset of both groups were also studied by hybrid capture-based DNA sequencing, probing not only for 479 cancer-related human genes, but also for polyoma and other viral sequences. Polyoma T-antigen-expressing tumors arose in 7 males and 4 females, at a median age of 66, and were aggressive, high-grade tumors with more than 1 variant morphologic pattern identified in 81% of cases, and a majority (73%) presenting at high stage category (>pT3). Diffuse polyoma T-antigen staining was seen in 91% of cases, with co-localization of aberrant p53 staining in 89%. Sequencing detected a lower number of deleterious mutations among T-antigen-expressing cases (average 1.62; 1/8 with TP53 mutation) compared to control cases (average 3.5, 2/4 with TP53 mutation). Only BK virus was detected with clonal integration and breakpoints randomly distributed across the human and viral genomes in 5/5 of the polyoma T-antigen-expressing carcinomas, and in none of the controls (0/4). In summary, these findings identify aggressive clinicopathologic features of polyoma T-antigen-expressing carcinomas, document BK as the strain involved, and associate BK viral integration with T-antigen expression and p53 aberrancy. While the apparent randomness of viral insertion sites is functionally unclear, the differing rates of mutations between T-antigen-expressing and control cases is intriguing.

RNA-Seq Analysis of Gene Expression, Viral Pathogen, and B-Cell/T-Cell Receptor Signatures in Complex Chronic Disease.

Background: Chronic fatigue syndrome (CFS) remains poorly understood. Although infections are speculated to trigger the syndrome, a specific infectious agent and underlying pathophysiological mechanism remain elusive. In a previous study, we described similar clinical phenotypes in CFS patients and alternatively diagnosed chronic Lyme syndrome (ADCLS) patients­individuals diagnosed with Lyme disease by testing from private Lyme specialty laboratories but who test negative by reference 2-tiered serologic analysis. Methods: Here, we performed blinded RNA-seq analysis of whole blood collected from 25 adults diagnosed with CFS and 13 ADCLS patients, comparing these cases to 25 matched controls and 11 patients with well-controlled systemic lupus erythematosus (SLE). Samples were collected at patient enrollment and not during acute symptom flares. RNA-seq data were used to study host gene expression, B-cell/T-cell receptor profiles (BCR/TCR), and potential viral infections. Results: No differentially expressed genes (DEGs) were found to be significant when CFS or ADCLS cases were compared to controls. Forty-two DEGs were found when SLE cases were compared to controls, consistent with activation of interferon signaling pathways associated with SLE disease. BCR/TCR repertoire analysis did not show significant differences between CFS and controls or ADCLS and controls. Finally, viral sequences corresponding to anelloviruses, human pegivirus 1, herpesviruses, and papillomaviruses were detected in RNA-seq data, but proportions were similar (P = .73) across all genus-level taxonomic categories. Conclusions: Our observations do not support a theory of transcriptionally mediated immune cell dysregulation in CFS and ADCLS, at least outside of periods of acute symptom flares.

Diagnosis of Fatal Human Case of St. Louis Encephalitis Virus Infection by Metagenomic Sequencing, California, 2016.

We used unbiased metagenomic next-generation sequencing to diagnose a fatal case of meningoencephalitis caused by St. Louis encephalitis virus in a patient from California in September 2016. This case is associated with the recent 2015-2016 reemergence of this virus in the southwestern United States.

Actionable diagnosis of neuroleptospirosis by next-generation sequencing.

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples.

Unbiased next-generation sequencing (NGS) approaches enable comprehensive pathogen detection in the clinical microbiology laboratory and have numerous applications for public health surveillance, outbreak investigation, and the diagnosis of infectious diseases. However, practical deployment of the technology is hindered by the bioinformatics challenge of analyzing results accurately and in a clinically relevant timeframe. Here we describe SURPI ("sequence-based ultrarapid pathogen identification"), a computational pipeline for pathogen identification from complex metagenomic NGS data generated from clinical samples, and demonstrate use of the pipeline in the analysis of 237 clinical samples comprising more than 1.1 billion sequences. Deployable on both cloud-based and standalone servers, SURPI leverages two state-of-the-art aligners for accelerated analyses, SNAP and RAPSearch, which are as accurate as existing bioinformatics tools but orders of magnitude faster in performance. In fast mode, SURPI detects viruses and bacteria by scanning data sets of 7-500 million reads in 11 min to 5 h, while in comprehensive mode, all known microorganisms are identified, followed by de novo assembly and protein homology searches for divergent viruses in 50 min to 16 h. SURPI has also directly contributed to real-time microbial diagnosis in acutely ill patients, underscoring its potential key role in the development of unbiased NGS-based clinical assays in infectious diseases that demand rapid turnaround times.

An ensemble strategy that significantly improves de novo assembly of microbial genomes from metagenomic next-generation sequencing data.

Next-generation sequencing (NGS) approaches rapidly produce millions to billions of short reads, which allow pathogen detection and discovery in human clinical, animal and environmental samples. A major limitation of sequence homology-based identification for highly divergent microorganisms is the short length of reads generated by most highly parallel sequencing technologies. Short reads require a high level of sequence similarities to annotated genes to confidently predict gene function or homology. Such recognition of highly divergent homologues can be improved by reference-free (de novo) assembly of short overlapping sequence reads into larger contigs. We describe an ensemble strategy that integrates the sequential use of various de Bruijn graph and overlap-layout-consensus assemblers with a novel partitioned sub-assembly approach. We also proposed new quality metrics that are suitable for evaluating metagenome de novo assembly. We demonstrate that this new ensemble strategy tested using in silico spike-in, clinical and environmental NGS datasets achieved significantly better contigs than current approaches.

Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing.

Metagenomic next-generation sequencing (NGS) was used to diagnose an unusual and fatal case of progressive encephalitis in an immunocompromised adult presenting at disease onset as bilateral hearing loss. The sequencing and confirmatory studies revealed neuroinvasive infection of the brain by an astrovirus belonging to a recently discovered VA/HMO clade.

Pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis.

Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma.

Compact CRISPR genetic screens enabled by improved guide RNA library cloning.

CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.

Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function.

Kidney disease is highly heritable; however, the causal genetic variants, the cell types in which these variants function, and the molecular mechanisms underlying kidney disease remain largely unknown. To identify genetic loci affecting kidney function, we performed a GWAS using multiple kidney function biomarkers and identified 462 loci. To begin to investigate how these loci affect kidney function, we generated single-cell chromatin accessibility (scATAC-seq) maps of the human kidney and identified candidate cis-regulatory elements (cCREs) for kidney podocytes, tubule epithelial cells, and kidney endothelial, stromal, and immune cells. Kidney tubule epithelial cCREs explained 58% of kidney function SNP-heritability and kidney podocyte cCREs explained an additional 6.5% of SNP-heritability. In contrast, little kidney function heritability was explained by kidney endothelial, stromal, or immune cell-specific cCREs. Through functionally informed fine-mapping, we identified putative causal kidney function variants and their corresponding cCREs. Using kidney scATAC-seq data, we created a deep learning model (which we named ChromKid) to predict kidney cell type-specific chromatin accessibility from sequence. ChromKid and allele specific kidney scATAC-seq revealed that many fine-mapped kidney function variants locally change chromatin accessibility in tubule epithelial cells. Enhancer assays confirmed that fine-mapped kidney function variants alter tubule epithelial regulatory element function. To map the genes which these regulatory elements control, we used CRISPR interference (CRISPRi) to target these regulatory elements in tubule epithelial cells and assessed changes in gene expression. CRISPRi of enhancers harboring kidney function variants regulated NDRG1 and RBPMS expression. Thus, inherited differences in tubule epithelial NDRG1 and RBPMS expression may predispose to kidney disease in humans. We conclude that genetic variants affecting tubule epithelial regulatory element function account for most SNP-heritability of human kidney function. This work provides an experimental approach to identify the variants, regulatory elements, and genes involved in polygenic disease.

Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function.

Kidney failure, the decrease of kidney function below a threshold necessary to support life, is a major cause of morbidity and mortality. We performed a genome-wide association study (GWAS) of 406,504 individuals in the UK Biobank, identifying 430 loci affecting kidney function in middle-aged adults. To investigate the cell types affected by these loci, we integrated the GWAS with human kidney candidate cis-regulatory elements (cCREs) identified using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). Overall, 56% of kidney function heritability localized to kidney tubule epithelial cCREs and an additional 7% to kidney podocyte cCREs. Thus, most heritable differences in adult kidney function are a result of altered gene expression in these two cell types. Using enhancer assays, allele-specific scATAC-seq and machine learning, we found that many kidney function variants alter tubule epithelial cCRE chromatin accessibility and function. Using CRISPRi, we determined which genes some of these cCREs regulate, implicating NDRG1, CCNB1 and STC1 in human kidney function.

Longitudinal comparison of the developing gut virome in infants and their mothers.

The human gut virome and its early life development are poorly understood. Prior studies have captured single-point assessments with the evolution of the infant virome remaining largely unexplored. We performed viral metagenomic sequencing on stool samples collected longitudinally from a cohort of 53 infants from age 2 weeks to 3 years (80.7 billion reads), and from their mothers (9.8 billion reads) to examine and compare viromes. The asymptomatic infant virome consisted of bacteriophages, nonhuman dietary/environmental viruses, and human-host viruses, predominantly picornaviruses. In contrast, human-host viruses were largely absent from the maternal virome. Previously undescribed, sequence-divergent vertebrate viruses were detected in the maternal but not infant virome. As infants aged, the phage component evolved to resemble the maternal virome, but by age 3, the human-host component remained dissimilar from the maternal virome. Thus, early life virome development is determined predominantly by dietary, infectious, and environmental factors rather than direct maternal acquisition.

Metagenomic Detection of Divergent Insect- and Bat-Associated Viruses in Plasma from Two African Individuals Enrolled in Blood-Borne Surveillance.

Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.