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1.
PLoS One ; 17(3): e0264855, 2022.
Article in English | MEDLINE | ID: covidwho-1736511

ABSTRACT

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).


Subject(s)
Bacteria/genetics , COVID-19/diagnosis , High-Throughput Nucleotide Sequencing/methods , RNA Viruses/genetics , Respiratory Tract Infections/diagnosis , SARS-CoV-2/genetics , Bacteria/isolation & purification , COVID-19/virology , Coronavirus/genetics , Coronavirus/isolation & purification , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/isolation & purification , Humans , Multiplex Polymerase Chain Reaction , Nanopores , Orthomyxoviridae/genetics , Orthomyxoviridae/isolation & purification , RNA Viruses/isolation & purification , RNA, Viral/chemistry , RNA, Viral/metabolism , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , SARS-CoV-2/isolation & purification
2.
Appl Environ Microbiol ; 87(23): e0144821, 2021 11 10.
Article in English | MEDLINE | ID: covidwho-1434881

ABSTRACT

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the coronavirus disease 2019 (COVID-19) pandemic to track disease burden through analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the health care system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven wastewater treatment plants (WTPs), collected from August 2020 to January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single-nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater, and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically important SARS-CoV-2 variants along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. IMPORTANCE Wastewater-based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the coronavirus disease 2019 (COVID-19) pandemic to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without adding burden to health care systems. In this study, we used metatranscriptomics and reverse transcription-droplet digital PCR (RT-ddPCR) to assay RNA viruses across Southern California wastewater from August 2020 to January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single-nucleotide variants through sequencing. Likewise, wastewater treatment plants (WTPs) harbored different viromes, and we detected other human pathogens, such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.


Subject(s)
RNA Viruses/isolation & purification , SARS-CoV-2/isolation & purification , Virome , Waste Water/virology , Wastewater-Based Epidemiological Monitoring , COVID-19/epidemiology , California , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , Polymerase Chain Reaction , RNA Viruses/classification , RNA Viruses/genetics , SARS-CoV-2/classification , SARS-CoV-2/genetics , Sequence Analysis, RNA
3.
Sci Rep ; 11(1): 15997, 2021 08 06.
Article in English | MEDLINE | ID: covidwho-1345579

ABSTRACT

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of 'sample-in and result-out' with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/µL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/µL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/µL with a total assay time of 15-30 min.


Subject(s)
COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , RNA Viruses/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , Equipment Design , Humans , Limit of Detection , Nucleic Acid Amplification Techniques/instrumentation , RNA Viruses/isolation & purification , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Saliva/virology
4.
Cell Rep Med ; 2(4): 100245, 2021 04 20.
Article in English | MEDLINE | ID: covidwho-1155662

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and variants has led to significant mortality. We recently reported that an RNA-targeting CRISPR-Cas13 system, called prophylactic antiviral CRISPR in human cells (PAC-MAN), offered an antiviral strategy against SARS-CoV-2 and influenza A virus. Here, we expand in silico analysis to use PAC-MAN to target a broad spectrum of human- or livestock-infectious RNA viruses with high specificity, coverage, and predicted efficiency. Our analysis reveals that a minimal set of 14 CRISPR RNAs (crRNAs) is able to target >90% of human-infectious viruses across 10 RNA virus families. We predict that a set of 5 experimentally validated crRNAs can target new SARS-CoV-2 variant sequences with zero mismatches. We also build an online resource (crispr-pacman.stanford.edu) to support community use of CRISPR-Cas13 for broad-spectrum RNA virus targeting. Our work provides a new bioinformatic resource for using CRISPR-Cas13 to target diverse RNA viruses to facilitate the development of CRISPR-based antivirals.


Subject(s)
CRISPR-Cas Systems/genetics , RNA Viruses/genetics , RNA, Guide/metabolism , COVID-19/pathology , COVID-19/virology , Humans , RNA Virus Infections/pathology , RNA Virus Infections/virology , RNA Viruses/isolation & purification , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Species Specificity
5.
PLoS One ; 16(3): e0248750, 2021.
Article in English | MEDLINE | ID: covidwho-1144198

ABSTRACT

BACKGROUND: Severe acute respiratory infection (SARI) results in a tremendous disease burden worldwide. Available research on active surveillance among hospitalized adult patients suffering from SARI in China is limited. This pilot study aimed to identify associated etiologies and describe the demographic, epidemiological and clinical profiles of hospitalized SARI patients aged over 16 years in Jinshan, Shanghai. METHODS: Active surveillance was conducted at 1 sentinel hospital in Jinshan district, Shanghai, from April 2017 to March 2018. Hospitalized SARI patients aged over 16 years old were enrolled, and nasopharyngeal swabs were collected within 24 hours of admission and tested for multiple respiratory viruses (including 18 common viruses) and Mycoplasma pneumoniae with real-time polymerase chain reaction. Demographic, epidemiological and clinical information was obtained from case report forms. RESULTS: In total, 397 SARI patients were enrolled; the median age was 68 years, and 194 (48.9%) patients were male. A total of 278 (70.0%) patients had at least one underlying chronic medical condition. The most frequent symptoms were cough (99.2%) and sputum production (88.4%). The median duration of hospitalization was 10 days. A total of 250 infection patients (63.0%) were positive for at least one pathogen, of whom 198 (49.9%) were positive for a single pathogen and 52 (13.1%) were positive for multiple pathogens. The pathogens identified most frequently were M. pneumoniae (23.9%, 95/397), followed by adenovirus (AdV) (11.6%, 46/397), influenza virus A/H3N2 (Flu A/H3N2) (11.1%, 44/397), human rhinovirus (HRhV) (8.1%, 32/397), influenza virus B/Yamagata (Flu B/Yamagata) (6.3%, 25/397), pandemic influenza virus A/H1N1 (Flu A/pH1N1) (4.0%, 16/397), parainfluenza virus (PIV) type 1 (2.0%, 8/397), human coronavirus (HCoV) type NL63 (2.0%, 8/397), HCoV 229E (1.5%, 6/397), HCoV HKU1 (1.5%, 6/397), PIV 3 (1.5%, 6/397), human metapneumovirus (HMPV) (1.5%, 6/397), PIV 4 (1.3%, 5/397), HCoV OC43 (1.0%, 4/397), influenza virus B/Victoria (Flu B/Victoria) (0.5%, 2/397), respiratory syncytial virus (RSV) type B (0.5%, 2/397), and human bocavirus (HBoV) (0.3%, 1/397). The seasonality of pathogen-confirmed SARI patients had a bimodal distribution, with the first peak in the summer and the second peak in the winter. Statistically significant differences were observed with respect to the rates of dyspnea, radiographically diagnosed pneumonia and the presence of at least one comorbidity in patients who were infected with only M. pneumoniae, AdV, HRhV, Flu A/H3N2, Flu A /pH1N1 or Flu B/Yamagata. The differences in the positivity rates of the above 6 pathogens among the different age groups were nonsignificant. CONCLUSIONS: M. pneumoniae, AdV and Flu A/H3N2 were the main pathogens detected in hospitalized SARI patients aged over 16 years old in Jinshan district, Shanghai. Our findings highlight the importance of sustained multipathogen surveillance among SARI patients in sentinel hospitals, which can provide useful information on SARI etiologies, epidemiology, and clinical characteristics.


Subject(s)
DNA Viruses/isolation & purification , Mycoplasma pneumoniae/isolation & purification , RNA Viruses/isolation & purification , Respiratory Tract Infections/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Anti-Bacterial Agents/therapeutic use , China , Cough/etiology , Female , Glucocorticoids/therapeutic use , Hospitals , Humans , Male , Middle Aged , Nasopharynx/microbiology , Nasopharynx/virology , Pilot Projects , Prognosis , Respiratory Tract Infections/drug therapy , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , Young Adult
6.
Sci Rep ; 11(1): 3209, 2021 02 05.
Article in English | MEDLINE | ID: covidwho-1065951

ABSTRACT

Viral co-infections occur in COVID-19 patients, potentially impacting disease progression and severity. However, there is currently no dedicated method to identify viral co-infections in patient RNA-seq data. We developed PACIFIC, a deep-learning algorithm that accurately detects SARS-CoV-2 and other common RNA respiratory viruses from RNA-seq data. Using in silico data, PACIFIC recovers the presence and relative concentrations of viruses with > 99% precision and recall. PACIFIC accurately detects SARS-CoV-2 and other viral infections in 63 independent in vitro cell culture and patient datasets. PACIFIC is an end-to-end tool that enables the systematic monitoring of viral infections in the current global pandemic.


Subject(s)
COVID-19/diagnosis , Coinfection/diagnosis , Deep Learning , RNA Virus Infections/diagnosis , RNA Viruses/isolation & purification , SARS-CoV-2/isolation & purification , COVID-19 Testing , Coinfection/virology , Coronaviridae/isolation & purification , Humans , Metapneumovirus/classification , Metapneumovirus/isolation & purification , Neural Networks, Computer , Orthomyxoviridae/classification , Orthomyxoviridae/isolation & purification , RNA Virus Infections/virology , RNA Viruses/classification , RNA-Seq , Rhinovirus/classification , Rhinovirus/isolation & purification , SARS-CoV-2/classification , Sensitivity and Specificity
7.
Biochemistry (Mosc) ; 86(3): 248-261, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1010982

ABSTRACT

Viral positive-sense RNA genomes evolve rapidly due to the high mutation rates during replication and RNA recombination, which allowing the viruses to acquire and modify genes for their adaptation. The size of RNA genome is limited by several factors, including low fidelity of RNA polymerases and packaging constraints. However, the 12-kb size limit is exceeded in the two groups of eukaryotic (+)RNA viruses - animal nidoviruses and plant closteroviruses. These virus groups have several traits in common. Their genomes contain 5'-proximal genes that are expressed via ribosomal frameshifting and encode one or two papain-like protease domains, membrane-binding domain(s), methyltransferase, RNA helicase, and RNA polymerase. In addition, some nidoviruses (i.e., coronaviruses) contain replication-associated domains, such as proofreading exonuclease, putative primase, nucleotidyltransferase, and endonuclease. In both nidoviruses and closteroviruses, the 3'-terminal part of the genome contains genes for structural and accessory proteins expressed via a nested set of coterminal subgenomic RNAs. Coronaviruses and closteroviruses have evolved to form flexuous helically symmetrical nucleocapsids as a mean to resolve packaging constraints. Since phylogenetic reconstructions of the RNA polymerase domains indicate only a marginal relationship between the nidoviruses and closteroviruses, their similar properties likely have evolved convergently, along with the increase in the genome size.


Subject(s)
Eukaryota/virology , Genome, Viral , RNA Viruses/chemistry , RNA Viruses/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Amino Acid Sequence , Animals , Biological Evolution , Humans , Open Reading Frames , RNA Viruses/isolation & purification , RNA Viruses/metabolism , RNA, Viral/metabolism
8.
Anal Methods ; 13(1): 34-55, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-962238

ABSTRACT

RNA-based viruses likely make up the highest pandemic threat among all known pathogens in about the last 100 years, since the Spanish Flu of 1918 with 50 M deaths up to COVID-19. Nowadays, an efficient and affordable testing strategy for such viruses have become the paramount target for the fields of virology and bioanalytical chemistry. The detection of the viruses (influenza, hepatitis, HIV, Zika, SARS, Ebola, SARS-CoV-2, etc.) and human antibodies to these viruses is described and tabulated in terms of the reported methods of detection, time to results, accuracy and specificity, if they are reported. The review is focused, but not limited to publications in the last decade. Finally, the limits of detection for each representative publication are tabulated by detection methods and discussed. These methods include PCR, lateral flow immunoassays, LAMP-based methods, ELISA, electrochemical methods (e.g., amperometry, voltammetry), fluorescence spectroscopy, AFM, SPR and SERS spectroscopy, silver staining and CRISPR-Cas based methods, bio-barcode detection, and resonance light scattering. The review is likely to be interesting for various scientists, and particularly helpful with information for establishing interdisciplinary research.


Subject(s)
Chemistry Techniques, Analytical/methods , Immunoassay/methods , RNA Viruses/isolation & purification , Antibodies, Viral/analysis , Data Accuracy , Humans , Limit of Detection , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
9.
J Biophotonics ; 13(10): e202000189, 2020 10.
Article in English | MEDLINE | ID: covidwho-627369

ABSTRACT

Several non-invasive Raman spectroscopy-based assays have been reported for rapid and sensitive detection of pathogens. We developed a novel statistical model for the detection of RNA viruses in saliva, based on an unbiased selection of a set of 65 Raman spectral features that mostly attribute to the RNA moieties, with a prediction accuracy of 91.6% (92.5% sensitivity and 88.8% specificity). Furthermore, to minimize variability and automate the downstream analysis of the Raman spectra, we developed a GUI-based analytical tool "RNA Virus Detector (RVD)." This conceptual framework to detect RNA viruses in saliva could form the basis for field application of Raman Spectroscopy in managing viral outbreaks, such as the ongoing COVID-19 pandemic. (http://www.actrec.gov.in/pi-webpages/AmitDutt/RVD/RVD.html).


Subject(s)
RNA Viruses/isolation & purification , Saliva/virology , Spectrum Analysis, Raman/methods , HEK293 Cells , Humans , User-Computer Interface
10.
Front Cell Infect Microbiol ; 10: 181, 2020.
Article in English | MEDLINE | ID: covidwho-266348

ABSTRACT

The availability of pathogen-specific treatment options for respiratory tract infections (RTIs) increased the need for rapid diagnostic tests. Besides, retrospective studies, improved lab-based detection methods and the intensified search for new viruses since the beginning of the twenty-first century led to the discovery of several novel respiratory viruses. Among them are human bocavirus (HBoV), human coronaviruses (HCoV-HKU1, -NL63), human metapneumovirus (HMPV), rhinovirus type C (RV-C), and human polyomaviruses (KIPyV, WUPyV). Additionally, new viruses like SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), novel strains of influenza virus A and B, and (most recently) SARS coronavirus 2 (SARS-CoV-2) have emerged. Although clinical presentation may be similar among different viruses, associated symptoms may range from a mild cold to a severe respiratory illness, and thus require a fast and reliable diagnosis. The increasing number of commercially available rapid point-of-care tests (POCTs) for respiratory viruses illustrates both the need for this kind of tests but also the problem, i.e., that the majority of such assays has significant limitations. In this review, we summarize recently published characteristics of POCTs and discuss their implications for the treatment of RTIs. The second key aspect of this work is a description of new and innovative diagnostic techniques, ranging from biosensors to novel portable and current lab-based nucleic acid amplification methods with the potential future use in point-of-care settings. While prototypes for some methods already exist, other ideas are still experimental, but all of them give an outlook of what can be expected as the next generation of POCTs.


Subject(s)
Communicable Diseases, Emerging/diagnosis , Communicable Diseases, Emerging/virology , Point-of-Care Testing , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/virology , Biosensing Techniques/methods , DNA Viruses/isolation & purification , Humans , Polymerase Chain Reaction/methods , RNA Viruses/isolation & purification
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