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1.
Swiss Med Wkly ; 152: w30202, 2022 06 20.
Article in English | MEDLINE | ID: covidwho-2202460

ABSTRACT

AIMS OF THE STUDY: Wastewater-based epidemiology has contributed significantly to the comprehension of the dynamics of the current COVID-19 pandemic. Its additional value in monitoring SARS-CoV-2 circulation in the population and identifying newly arising variants independently of diagnostic testing is now undisputed. As a proof of concept, we report here correlations between SARS-CoV-2 detection in wastewater and the officially recorded COVID-19 case numbers, as well as the validity of such surveillance to detect emerging variants, exemplified by the detection of the B.1.1.529 variant Omicron in Basel, Switzerland. METHODS: From July 1 to December 31, 2021, wastewater samples were collected six times a week from the inflow of the local wastewater treatment plant that receives wastewater from the catchment area of the city of Basel, Switzerland, comprising 273,075 inhabitants. The number of SARS-CoV-2 RNA copies was determined by reverse transcriptase-quantitative PCR. Spearman's rank correlation coefficients were calculated to determine correlations with the median seven-day incidence of genome copies per litre of wastewater and official case data. To explore delayed correlation effects between the seven-day median number of genome copies/litre wastewater and the median seven-day incidence of SARS-CoV-2 cases, time-lagged Spearman's rank correlation coefficients were calculated for up to 14 days. RNA extracts from daily wastewater samples were used to genotype circulating SARS-CoV-2 variants by next-generation sequencing. RESULTS: The number of daily cases and the median seven-day incidence of SARS-CoV-2 infections in the catchment area showed a high correlation with SARS-CoV-2 measurements in wastewater samples. All correlations between the seven-day median number of genome copies/litre wastewater and the time-lagged median seven-day incidence of SARS-CoV-2 cases were significant (p<0.001) for the investigated lag of up to 14 days. Correlation coefficients declined constantly from the maximum of 0.9395 on day 1 to the minimum of 0.8016 on day 14. The B.1.1.529 variant Omicron was detected in wastewater samples collected on November 21, 2021, before its official acknowledgement in a clinical sample by health authorities. CONCLUSIONS: In this proof-of-concept study, wastewater-based epidemiology proved a reliable and sensitive surveillance approach, complementing routine clinical testing for mapping COVID-19 pandemic dynamics and observing newly circulating SARS-CoV-2 variants.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/epidemiology , Humans , Pandemics , RNA, Viral/genetics , SARS-CoV-2/genetics , Switzerland/epidemiology , Waste Water/analysis
2.
Prague Med Rep ; 123(4): 250-257, 2022.
Article in English | MEDLINE | ID: covidwho-2145507

ABSTRACT

The SARS-CoV-2 viral load in a respiratory sample can be inversely quantified using the cycle threshold (Ct), defined as the number of amplification cycles required to detect the viral genome in a quantitative PCR assay using reverse transcriptase (RT-qPCR). It may be classified as high (Ct < 25), intermediate (25-30) and low (Ct > 30). We describe the clinical course of 3 patients with haematological neoplasms who contracted COVID-19. None of them had been vaccinated. Firstly, a 22-year-old male with a refractory acute lymphoblastic leukaemia experienced an oligosymptomatic COVID-19 and had a Ct of 23 with an ascending curve. Another male, aged 23, had recently begun treatment for a promyelocytic leukaemia. He had a subacute course with high oxygen requirements. His Ct dropped from 28, when he only experienced fever, to 14.8, during the most critical period and on the edge of ventilatory support. Viral clearance was documented 126 days after the beginning of the symptoms. Finally, a 60-year-old male had received rituximab as maintenance therapy for a follicular lymphoma 3 months before contracting COVID-19. He had a fulminant course and required mechanical ventilation a few days later. We highlight the association between the course of CoViD-19 and the Ct. Viral shedding was longer than in immunocompetent hosts.


Subject(s)
COVID-19 , Hematologic Neoplasms , Neoplasms , Humans , Male , Young Adult , Adult , Middle Aged , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2 , Hematologic Neoplasms/complications
3.
Viruses ; 14(11)2022 Nov 04.
Article in English | MEDLINE | ID: covidwho-2143699

ABSTRACT

Small molecular nucleic acid drugs produce antiviral effects by activating pattern recognition receptors (PRRs). In this study, a small molecular nucleotide containing 5'triphosphoric acid (5'PPP) and possessing a double-stranded structure was designed and named nCoV-L. nCoV-L was found to specifically activate RIG-I, induce interferon responses, and inhibit duplication of four RNA viruses (Human enterovirus 71, Human poliovirus 1, Human coxsackievirus B5 and Influenza A virus) in cells. In vivo, nCoV-L quickly induced interferon responses and protected BALB/c suckling mice from a lethal dose of the enterovirus 71. Additionally, prophylactic administration of nCoV-L was found to reduce mouse death and relieve morbidity symptoms in a K18-hACE2 mouse lethal model of SARS-CoV-2. In summary, these findings indicate that nCoV-L activates RIG-I and quickly induces effective antiviral signals. Thus, it has potential as a broad-spectrum antiviral drug.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mice , Animals , DEAD-box RNA Helicases/genetics , RNA, Viral/genetics , Cell Line , DEAD Box Protein 58 , Mice, Inbred BALB C , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Interferons
4.
PLoS One ; 17(11): e0278061, 2022.
Article in English | MEDLINE | ID: covidwho-2140683

ABSTRACT

Contaminated surfaces are one of the ways that coronavirus disease 2019 (COVID-19) may be transmitted. SARS-CoV-2 can be detected on environmental surfaces; however, few environmental sampling studies have been conducted in nonclinical settings. The objective of this study was to detect SARS-CoV-2 RNA on environmental surfaces in public areas in Las Vegas, Nevada. In total, 300 surface samples were collected from high-touch surfaces from high-congregate public locations and from a public health facility (PHF) that was visited by COVID-19 patients. Environmental samples were analyzed with quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) using SARS-CoV-2 specific primers and probes for three target genes. Results showed that 31 out of 300 (10.3%) surface samples tested positive for SARS-CoV-2, 24 at the PHF and 7 in high-congregate public locations. Concentrations ranged from 102 to 106 viral particles per 3 ml sample on a wide variety of materials. The data also showed that the N gene assay had greater sensitivity compared to the S and ORF gene assays. Besides frequently touched surfaces, SARS-CoV-2 was detected in restrooms, on floors and surfaces in contact with floors, as well as in a mop water sample. The results of this study describe the extent and distribution of environmental SARS-CoV-2 contamination in public areas in Las Vegas, Nevada. A method using the N gene PCR assay was developed for SARS-CoV-2 environmental monitoring in public areas. Environmental monitoring with this method can determine the specific sites of surface contamination in the community and may be beneficial for prevention of COVID-19 indirect transmission, and evaluation and improvement of infection control practices in public areas, public health facilities, universities, and businesses.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , RNA, Viral/genetics , RNA, Viral/analysis , COVID-19/epidemiology , Specimen Handling , DNA Primers
5.
PLoS One ; 17(11): e0277881, 2022.
Article in English | MEDLINE | ID: covidwho-2140668

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes the global COVID-19 pandemic. Limited studies have been performed on various types of disinfectants utilized to control the spread of this highly contagious virus. This study aimed to investigate the inactivation of SARS-CoV-2 using compressed sodium chloride (CSC) surface. A real-time reverse transcriptase quantitative PCR (RT-qPCR) assay was used to evaluate the effectiveness of CSC on the disintegration of viral RNA in a time dependent manner. The effects of CSC on viral infectivity were determined using a TCID50 assay of a surrogate virus, hCoV-229E, in MRC-5 cell culture. The results demonstrated that CSC achieved a 2 to 3- log10 reduction of viral genomic RNA for a laboratory strain of hCoV-229E, and clinical samples of hCoV-229E and hCoV-OC43. A 3 to 4-log10 reduction was observed for SARS-CoV-2 (RdRp and E gene) suggesting that a CSC surface could effectively disintegrate the SARS-CoV-2 RNA genome. CSC was observed to have a 6 log10 inactivation of infectious hCoV-229E using cell culture after 5 minutes of exposure compared to the control, indicating good disinfection efficacy of a CSC surface against virus.


Subject(s)
COVID-19 , Coronavirus 229E, Human , Humans , SARS-CoV-2 , RNA, Viral/genetics , RNA, Viral/analysis , Sodium Chloride/pharmacology , Pandemics
6.
Clin Lab Med ; 42(2): 237-248, 2022 06.
Article in English | MEDLINE | ID: covidwho-2130431

ABSTRACT

Reverse transcription-polymerase chain reaction (RT-PCR) tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), are approved for qualitative use. The cycle threshold (Ct) value reflects the concentration of viral RNA in the sample, with lower Ct values indicating higher levels of RNA. Caregivers may wish to use the Ct value to determine the progression of infection, how severe the infection will be, and whether the patient can transmit the virus. Variability of Ct values and the data supporting these uses should be considered when deciding whether and how to use Ct values in clinical care.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2/genetics
7.
Anal Chim Acta ; 1234: 340533, 2022 Nov 22.
Article in English | MEDLINE | ID: covidwho-2129675

ABSTRACT

The emerging pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) critically challenges early and accurate virus diagnoses. However, the current gold standard for SARS-CoV-2 detection, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), has reportedly failed to detect low-viral loads. One compound, graphene oxide (GO), which adsorbs single-stranded DNA (ssDNA), has been widely applied in molecular pathogen detection. This study presents a highly sensitive GO-multiplex qPCR method for simultaneous detection of two SARS-CoV-2 genes (RdRP and E) and one reference gene (RNase P). In a GO-multiplex qPCR system, GO pre-absorbs each forward primer to form specific GO-forward primer composites before entering the amplification system. Target gene amplification is confined within the primer-enriched composites, thus, improving the sensitivity of the assay. Compared to conventional multiplex qPCR, GO-multiplex qPCR reduces the limit of detection by 10-fold to 10 copies/reaction. Hence, the GO-multiplex qPCR assay can be effectively used for SARS-CoV-2 detection.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19 Testing , Clinical Laboratory Techniques/methods , COVID-19/diagnosis , RNA, Viral/genetics , RNA, Viral/analysis , Sensitivity and Specificity
8.
Sci Data ; 9(1): 713, 2022 11 18.
Article in English | MEDLINE | ID: covidwho-2133500

ABSTRACT

Nationwide, wastewater-based monitoring was newly established in Scotland to track the levels of SARS-CoV-2 viral RNA shed into the sewage network, during the COVID-19 pandemic. We present a curated, reference dataset produced by this national programme, from May 2020 to February 2022. Viral levels were analysed by RT-qPCR assays of the N1 gene, on RNA extracted from wastewater sampled at 162 locations. Locations were sampled up to four times per week, typically once or twice per week, and in response to local needs. We report sampling site locations with geographical coordinates, the total population in the catchment for each site, and the information necessary for data normalisation, such as the incoming wastewater flow values and ammonia concentration, when these were available. The methodology for viral quantification and data analysis is briefly described, with links to detailed protocols online. These wastewater data are contributing to estimates of disease prevalence and the viral reproduction number (R) in Scotland and in the UK.


Subject(s)
COVID-19 , RNA, Viral , Humans , Pandemics , RNA, Viral/genetics , SARS-CoV-2 , Waste Water , Scotland
9.
PLoS One ; 17(11): e0276697, 2022.
Article in English | MEDLINE | ID: covidwho-2112663

ABSTRACT

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC-5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. In addition, we characterised the viral transcriptome in infected Calu-3 lung epithelial cells at 48 hours post infection. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing using an Oxford nanopore device. This platform yielded over 450,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine the relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We examined the frequency of polycistronic readthrough mRNAs were generated and assessed the length of the polyadenylated tails for each group of transcripts. We show a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not uniform. The polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host polyadenylation machinery and broadly declined in length for most transcripts as the infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails less than 20 nucleotides is less for N, SH and G transcripts in both cell lines compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates within and between the cell lines.


Subject(s)
Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , Humans , RNA, Messenger/genetics , RNA, Viral/genetics , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus Infections/genetics , Sequence Analysis, RNA
10.
Viruses ; 14(10)2022 10 06.
Article in English | MEDLINE | ID: covidwho-2110268

ABSTRACT

RNA-dependent RNA polymerases (RdRPs) represent a distinctive yet versatile class of nucleic acid polymerases encoded by RNA viruses for the replication and transcription of their genome. The structure of the RdRP is comparable to that of a cupped right hand consisting of fingers, palm, and thumb subdomains. Despite the presence of a common structural core, the RdRPs differ significantly in the mechanistic details of RNA binding and polymerization. The present review aims at exploring these incongruities in light of recent structural studies of RdRP complexes with diverse cofactors, RNA moieties, analogs, and inhibitors.


Subject(s)
Nucleic Acids , RNA Viruses , RNA-Dependent RNA Polymerase/genetics , RNA Viruses/genetics , DNA-Directed RNA Polymerases , RNA , RNA, Viral/genetics
11.
Viruses ; 14(11)2022 Nov 21.
Article in English | MEDLINE | ID: covidwho-2123866

ABSTRACT

Recombination and mutation of viral genomes represent major mechanisms for viral evolution and, in many cases, moderate pathogenicity. Segmented genome viruses frequently undergo reassortment of the genome via multiple infection of host organisms, with influenza and reoviruses being well-known examples. Specifically, major genomic shifts mediated by reassortment are responsible for radical changes in the influenza antigenic determinants that can result in pandemics requiring rapid preventative responses by vaccine modifications. In contrast, smaller mutational changes brought about by the error-prone viral RNA polymerases that, for the most part, lack a replication base mispairing editing function produce small mutational changes in the RNA genome during replication. Referring again to the influenza example, the accumulated mutations-known as drift-require yearly vaccine updating and rapid worldwide distribution of each new formulation. Coronaviruses with a large positive-sense RNA genome have long been known to undergo intramolecular recombination likely mediated by copy choice of the RNA template by the viral RNA polymerase in addition to the polymerase-based mutations. The current SARS-CoV-2 origin debate underscores the importance of understanding the plasticity of viral genomes, particularly the mechanisms responsible for intramolecular recombination. This review describes the use of the cystovirus bacteriophage as an experimental model for recombination studies in a controlled manner, resulting in the development of a model for intramolecular RNA genome alterations. The review relates the sequence of experimental studies from the laboratory of Leonard Mindich, PhD at the Public Health Research Institute-then in New York City-and covers a period of approximately 12 years. Hence, this is a historical scientific review of research that has the greatest relevance to current studies of emerging RNA virus pathogens.


Subject(s)
COVID-19 , Cystoviridae , Influenza, Human , Humans , Cystoviridae/genetics , SARS-CoV-2 , RNA, Viral/genetics , Recombination, Genetic
12.
J Infect Dis ; 226(10): 1743-1752, 2022 Nov 11.
Article in English | MEDLINE | ID: covidwho-2121302

ABSTRACT

BACKGROUND: Laboratory screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key mitigation measure to avoid the spread of infection among recruits starting basic combat training in a congregate setting. Because viral nucleic acid can be detected persistently after recovery, we evaluated other laboratory markers to distinguish recruits who could proceed with training from those who were infected. METHODS: Recruits isolated for coronavirus disease 2019 (COVID-19) were serially tested for SARS-CoV-2 subgenomic ribonucleic acid (sgRNA), and viral load (VL) by reverse-transcriptase polymerase chain reaction (RT-PCR), and for anti- SARS-CoV-2. Cluster and quadratic discriminant analyses of results were performed. RESULTS: Among 229 recruits isolated for COVID-19, those with a RT-PCR cycle threshold >30.49 (sensitivity 95%, specificity 96%) or having sgRNA log10 RNA copies/mL <3.09 (sensitivity and specificity 96%) at entry into isolation were likely SARS-CoV-2 uninfected. Viral load >4.58 log10 RNA copies/mL or anti-SARS-CoV-2 signal-to-cutoff ratio <1.38 (VL: sensitivity and specificity 93%; anti-SARS-CoV-2: sensitivity 83%, specificity 79%) had comparatively lower sensitivity and specificity when used alone for discrimination of infected from uninfected. CONCLUSIONS: Orthogonal laboratory assays used in combination with RT-PCR may have utility in determining SARS-CoV-2 infection status for decisions regarding isolation.


Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , SARS-CoV-2 , COVID-19 Testing , Sensitivity and Specificity , RNA , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction
13.
Front Immunol ; 13: 1008084, 2022.
Article in English | MEDLINE | ID: covidwho-2119705

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic, resulting in great fatalities around the world. Although the antiviral roles of RNA interference (RNAi) have been well studied in plants, nematodes and insects, the antiviral roles of RNAi in mammalians are still debating as RNAi effect is suspected to be suppressed by interferon (IFN) signaling pathways in most cell types. To determine the role of RNAi in mammalian resistance to SARS-CoV-2, we studied the profiling of host small RNAs and SARS-CoV-2 virus-derived small RNAs (vsRNAs) in the early infection stages of Vero cells, an IFN-deficient cell line. We found that host microRNAs (miRNAs) were dysregulated upon SARS-CoV-2 infection, resulting in downregulation of microRNAs playing antiviral functions and upregulation of microRNAs facilitating viral proliferations. Moreover, vsRNA peaked at 22 nt at negative strand but not the positive strand of SARS-CoV-2 and formed successive Dicer-spliced pattern at both strands. Similar characteristics of vsRNAs were observed in IFN-deficient cell lines infected with Sindbis and Zika viruses. Together, these findings indicate that host cell may deploy RNAi pathway to combat SARS-CoV-2 infection in IFN-deficient cells, informing the alternative antiviral strategies to be developed for patients or tissues with IFN deficiency.


Subject(s)
COVID-19 , MicroRNAs , Zika Virus Infection , Zika Virus , Chlorocebus aethiops , Animals , Humans , Vero Cells , SARS-CoV-2/genetics , RNA, Viral/genetics , COVID-19/genetics , MicroRNAs/genetics , Antiviral Agents , Mammals
14.
Sci Rep ; 12(1): 19416, 2022 Nov 12.
Article in English | MEDLINE | ID: covidwho-2119170

ABSTRACT

The current COVID-19 pandemic outbreak poses a serious threat to public health, demonstrating the critical need for the development of effective and reproducible detection tests. Since the RT-qPCR primers are highly specific and can only be designed based on the known sequence, mutation sensitivity is its limitation. Moreover, the mutations in the severe acute respiratory syndrome ß-coronavirus (SARS-CoV-2) genome led to new highly transmissible variants such as Delta and Omicron variants. In the case of mutation, RT-qPCR primers cannot recognize and attach to the target sequence. This research presents an accurate dual-platform DNA biosensor based on the colorimetric assay of gold nanoparticles and the surface-enhanced Raman scattering (SERS) technique. It simultaneously targets four different regions of the viral genome for detection of SARS-CoV-2 and its new variants prior to any sequencing. Hence, in the case of mutation in one of the target sequences, the other three probes could detect the SARS-CoV-2 genome. The method is based on visible biosensor color shift and a locally enhanced electromagnetic field and significantly amplified SERS signal due to the proximity of Sulfo-Cyanine 3 (Cy3) and AuNPs intensity peak at 1468 cm-1. The dual-platform DNA/GO/AuNP biosensor exhibits high sensitivity toward the viral genome with a LOD of 0.16 ng/µL. This is a safe point-of-care, naked-eye, equipment-free, and rapid (10 min) detection biosensor for diagnosing COVID-19 cases at home using a nasopharyngeal sample.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Humans , SARS-CoV-2/genetics , Gold , Pandemics , COVID-19/diagnosis , Biosensing Techniques/methods , Genome, Viral/genetics , DNA , RNA, Viral/genetics
15.
BMC Genomics ; 23(1): 755, 2022 Nov 17.
Article in English | MEDLINE | ID: covidwho-2116898

ABSTRACT

BACKGROUND: Since inception of the COVID-19 pandemic, early detection and isolation of positive cases is one of the key strategies to restrict disease transmission. Real time reverse transcription polymerase chain reaction (qRTPCR) has been the mainstay of diagnosis. Most of the qRTPCR kits were designed against the target genes of original strain of SARS-CoV-2. However, with the emergence of variant strains of SARS-CoV-2, sensitivity of the qRTPCR assays has reportedly reduced. In view of this, it is critical to continuously monitor the performance of the qRTPCR kits in the backdrop of variant strains of SARS-CoV-2. Real world monitoring of assay performance is challenging. Therefore, we developed a two-step in-silico screening process for evaluating the performance of various qRTPCR kits used in India. RESULTS: We analysed 73 qRT-PCR kits marketed in India, against the two SARS-CoV-2 VoCs. Sequences of both Delta (B.1.617.2) and Omicron (B.1.1.529) VoCs submitted to GISAID within a specific timeframe were downloaded, clustered to identify unique sequences and aligned with primer and probe sequences. Results were analysed following a two-step screening process. Out of 73 kits analysed, seven were unsatisfactory for detection of both Delta and Omicron VoCs, 10 were unsatisfactory for Delta VoC whereas 2 were unsatisfactory for only Omicron VoC. CONCLUSION: Overall, we have developed a useful screening process for evaluating the performance of qRTPCR assays against Delta and Omicron VoCs of SARS-CoV-2 which can be used for detecting SARS-CoV-2 VoCs that may emerge in future and can also be redeployed for other evolving pathogens of public health importance.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Pandemics , RNA, Viral/genetics , RNA, Viral/analysis , Sensitivity and Specificity , COVID-19/diagnosis , COVID-19/epidemiology
16.
Nat Commun ; 13(1): 6716, 2022 Nov 16.
Article in English | MEDLINE | ID: covidwho-2116835

ABSTRACT

The unprecedented emergence and spread of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, underscores the need for diagnostic and therapeutic technologies that can be rapidly tailored to novel threats. Here, we show that site-specific RNA endonuclease XNAzymes - artificial catalysts composed of single-stranded synthetic xeno-nucleic acid oligonucleotides (in this case 2'-deoxy-2'-fluoro-ß-D-arabino nucleic acid) - may be designed, synthesised and screened within days, enabling the discovery of a range of enzymes targeting SARS-CoV-2 ORF1ab, ORF7b, spike- and nucleocapsid-encoding RNA. Three of these are further engineered to self-assemble into a catalytic nanostructure with enhanced biostability. This XNA nanostructure is capable of cleaving genomic SARS-CoV-2 RNA under physiological conditions, and when transfected into cells inhibits infection with authentic SARS-CoV-2 virus by RNA knockdown. These results demonstrate the potential of XNAzymes to provide a platform for the rapid generation of antiviral reagents.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , RNA, Viral/genetics , Pandemics , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use
17.
Clin Infect Dis ; 75(10): 1698-1705, 2022 Nov 14.
Article in English | MEDLINE | ID: covidwho-2116480

ABSTRACT

The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of US Food and Drug Administration emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, that is, the presence of replicable virus. As the number of tests conducted increased, persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA positivity by reverse-transcription polymerase chain reaction (RT-PCR) in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike, nucleocapsid, membrane, envelope, and ORF8. Viral RNA abundances calculated from these assays were compared with antigen presence, self-reported symptoms, and culture outcome (virus isolation) using samples from a 14-day longitudinal household transmission study. By characterizing the clinical and molecular dynamics of infection, we show that sgRNA detection has higher predictive value for culture outcome compared to detection of gRNA alone. Our findings suggest that sgRNA presence correlates with active infection and may help identify individuals shedding culturable virus.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , RNA, Viral/genetics , RNA, Viral/analysis , Self Report , Longitudinal Studies , RNA, Guide , COVID-19/diagnosis
18.
Viruses ; 14(11)2022 Nov 21.
Article in English | MEDLINE | ID: covidwho-2116086

ABSTRACT

Background: The transmissible capacity and toxicity of SARS-CoV-2 variants are continually changing. We report here the follow-up study of hospitalized COVID-19 patients from 2020 to 2022. It is known that the PCR diagnosis for hospitalized patients sometimes causes confusion because of the incompatibility between their diagnosis and symptoms. We applied our sugar chain-immobilized gold-nanoparticles for the extraction and partial purification of RNA from specimens for quantitative RT-PCR assay and evaluated whether the results correlate with patients' symptoms. Methods and Results: Saliva specimens were taken from hospitalized patients with mild or moderate symptoms every early morning. At the time of RT-PCR diagnosis, two methods for the extraction and partial purification of RNA from the specimen were performed: a commonly used Boom (Qiagen) method and our original sugar chain-immobilized gold nanoparticle (SGNP) method. For symptoms, body temperature and oxygen saturation (SpO2) of patients were monitored every 4 h. Conclusions: It was clear that patients infected with the Delta variant needed more time to recover than those with the Omicron variant, and that the SGNP method showed more realistic correlation with the symptoms of patients compared with the common Qiagen method.


Subject(s)
COVID-19 , Metal Nanoparticles , Humans , Reverse Transcriptase Polymerase Chain Reaction , Gold , SARS-CoV-2/genetics , Sugars , Follow-Up Studies , COVID-19/diagnosis , RNA, Viral/genetics , RNA, Viral/analysis , Sensitivity and Specificity , Carbohydrates
19.
J Appl Genet ; 63(3): 583-585, 2022 09.
Article in English | MEDLINE | ID: covidwho-1824859
20.
Proc Biol Sci ; 289(1987): 20221747, 2022 Nov 30.
Article in English | MEDLINE | ID: covidwho-2115857

ABSTRACT

The raw material for viral evolution is provided by intra-host mutations occurring during replication, transcription or post-transcription. Replication and transcription of Coronaviridae proceed through the synthesis of negative-sense 'antigenomes' acting as templates for positive-sense genomic and subgenomic RNA. Hence, mutations in the genomes of SARS-CoV-2 and other coronaviruses can occur during (and after) the synthesis of either negative-sense or positive-sense RNA, with potentially distinct patterns and consequences. We explored for the first time the mutational spectrum of SARS-CoV-2 (sub)genomic and anti(sub)genomic RNA. We use a high-quality deep sequencing dataset produced using a quantitative strand-aware sequencing method, controlled for artefacts and sequencing errors, and scrutinized for accurate detection of within-host diversity. The nucleotide differences between negative- and positive-sense strand consensus vary between patients and do not show dependence on age or sex. Similarities and differences in mutational patterns between within-host minor variants on the two RNA strands suggested strand-specific mutations or editing by host deaminases and oxidative damage. We observe generally neutral and slight negative selection on the negative strand, contrasting with purifying selection in ORF1a, ORF1b and S genes of the positive strand of the genome.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , RNA, Viral/genetics , Genome, Viral , Mutation , Genomics
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