RESUMO
Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.
Assuntos
Hepatite B Crônica , Neoplasias Hepáticas , Humanos , Vírus da Hepatite B/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , RNA , Hepatite B Crônica/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamento farmacológico , Replicação ViralRESUMO
BACKGROUND: Powassan virus (POWV) is an emerging cause of severe encephalitis; very little is known about human pathogenicity due to challenges in diagnosis and viral RNA recovery. We present 3 patients with fatal encephalitis due to POWV lineage II (deer tick virus). METHODS: We obtained 27 unique samples, including from brain biopsy and autopsy, and used metagenomic sequencing, quantitative reverse transcriptase polymerase chain reaction, and a newly developed CRISPR-based diagnostic assay to perform the first detailed characterization of POWV compartmentalization and genomics between and within human subjects. RESULTS: In all 3 patients, imaging and histopathology findings were notable for profound cerebellar involvement. All patients were initially diagnosed with POWV by metagenomic sequencing, and 2 of the 3 had negative clinical testing by serology. We detected POWV RNA in 13 clinical samples; levels were highest in the cerebellum, and there was very little involvement of peripheral tissue. We assembled complete POWV genomes from 8 samples, providing unique information about the strains of POWV lineage II (deer tick virus) that infect humans. CONCLUSIONS: We demonstrate the utility of molecular assays for detecting POWV infection, including in seronegative patients, and nominate viral genomic features that may relate to human infection and neuropathogenicity. The cerebellum was identified as a key target POWV in fatal infection, by radiological and histopathological findings as well as molecular testing.
RESUMO
The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas Analíticas Microfluídicas/métodos , Viroses/diagnóstico , Viroses/virologia , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Farmacorresistência Viral/genética , Genoma Viral/genética , HIV/classificação , HIV/genética , HIV/isolamento & purificação , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , RNA Guia de Cinetoplastídeos/genética , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
Assuntos
Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/análise , Vírus da Dengue/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , Ribonucleases/química , Zika virus/isolamento & purificação , Bactérias/patogenicidade , DNA Tumoral Circulante/análise , DNA Tumoral Circulante/genética , Dengue/diagnóstico , Vírus da Dengue/genética , Humanos , Mutação , Neoplasias/genética , Clivagem do RNA , RNA Viral/genética , Zika virus/genética , Infecção por Zika virus/diagnósticoRESUMO
Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of â¼ 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log2 fold change ≥ 1, false discovery rate ≤ 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.