ABSTRACT
BACKGROUND: Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA. RESULTS: Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. CONCLUSION: TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.
Subject(s)
COVID-19 , Papillomavirus Infections , Humans , Multiplex Polymerase Chain Reaction/methods , Reproducibility of Results , SARS-CoV-2/genetics , Papillomaviridae/genetics , Genomics , High-Throughput Nucleotide Sequencing/methods , DNA, Viral/genetics , COVID-19 TestingABSTRACT
High-risk human papillomavirus (hr-HPV) testing for primary cervical precancer screening offers an opportunity to improve screening in low-middle income countries (LMICs). This study aimed to compare the analytic performances of the AmpFire and MA-6000 platforms for hr-HPV DNA testing in three groups of women screened for hr-HPV types in Ghana: group 1 with 33 GeneXpert-archived ThinPrep/liquid-based samples subjected to both tests, group 2 with 50 AmpFire-archived dry brush samples subjected to MA-6000 testing, and group 3 involving 143 cotton swab samples simultaneously subjected to both tests without archiving. The overall agreement rates were 73 %, 92 %, and 84 %, for groups 1-3, respectively, and 84 % (95 % CI, 78.6-88.6) for the entire group. Neither AmpFire nor MA-6000 was more likely to test hr-HPV positive in all three groups and the combined group. Group 1 showed fair agreement without statistical significance (κ = 0.224, 95 % CI, -0.118 to 0.565), while group 3 showed significant moderate agreement (κ = 0.591, 95% CI, 0.442-0.741). Group 2 showed an almost perfect significant level of agreement (κ = 0.802; 95 % CI, 0.616-0.987). Thus, both platforms showed statistically significant moderate to near-perfect agreement for detecting hr-HPV in cervicovaginal samples, with variation according to archiving conditions and duration between sample collection and retesting. For LMICs using these platforms for COVID-19 testing, as the COVID-19 pandemic subsides, the platforms can become available for running other tests such as hr-HPV DNA testing for cervical precancer screening.
Subject(s)
COVID-19 , Papillomavirus Infections , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Female , Humans , Human Papillomavirus Viruses , Papillomavirus Infections/diagnosis , Papillomavirus Infections/epidemiology , COVID-19 Testing , Pandemics , COVID-19/diagnosis , Uterine Cervical Dysplasia/diagnosis , Polymerase Chain Reaction , Papillomaviridae/genetics , Uterine Cervical Neoplasms/diagnosis , Early Detection of Cancer , DNA, Viral/genetics , DNA, Viral/analysis , Sensitivity and SpecificityABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays have been an emerging diagnostic technology for pathogen diagnosis. In this work, we developed a polydisperse droplet digital CRISPR-Cas-based assay (PddCas) for the rapid and ultrasensitive amplification-free detection of viral DNA/RNA with minimum instruments. LbaCas12a and LbuCas13a were used for the direct detection of viral DNA and RNA, respectively. The reaction mixtures were partitioned with a common vortex mixer to generate picoliter-scale polydisperse droplets in several seconds. The limit of detection (LoD) for the target DNA and RNA is approximately 100 aM and 10 aM, respectively, which is about 3 × 104-105 fold more sensitive than corresponding bulk CRISPR assays. We applied the PddCas to successfully detect severe acute respiratory syndrome coronavirus (SARS-CoV-2) and human papillomavirus type 18 (HPV 18) in clinical samples. For the 23 HPV 18-suspected cervical epithelial cell samples and 32 nasopharyngeal swabs for SARS-CoV-2, 100% sensitivity and 100% specificity were demonstrated. The dual-gene virus detection with PddCas was also established and verified. Therefore, PddCas has potential for point-of-care application and is envisioned to be readily deployed for frequent testing as part of an integrated public health surveillance program.
Subject(s)
COVID-19 , Papillomavirus Infections , Humans , DNA, Viral/genetics , RNA, Viral/genetics , CRISPR-Cas Systems/genetics , SARS-CoV-2/genetics , Human papillomavirus 18ABSTRACT
Drug resistance caused by mutations is a public health threat for existing and emerging viral diseases. A wealth of evidence about these mutations and their clinically associated phenotypes is scattered across the literature, but a comprehensive perspective is usually lacking. This work aimed to produce a clinically relevant view for the case of Hepatitis B virus (HBV) mutations by combining a chronic HBV clinical study with a compendium of genetic mutations systematically gathered from the scientific literature. We enriched clinical mutation data by systematically mining 2,472,725 scientific articles from PubMed Central in order to gather information about the HBV mutational landscape. By performing this analysis, we were able to identify mutational hotspots for each HBV genotype (A-E) and gene (C, X, P, S), as well as the location of disulfide bonds associated with these mutations. Through a modelling study, we also identified a mutation position common in both the clinical data and the literature that is located at the binding pocket for a known anti-HBV drug, namely entecavir. The results of this novel approach show the potential of integrated analyses to assist in the development of new drugs for viral diseases that are more robust to resistance. Such analyses should be of particular interest due to the increasing importance of viral resistance in established and emerging viruses, such as for newly developed drugs against SARS-CoV-2.
Subject(s)
COVID-19 Drug Treatment , Hepatitis B, Chronic , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , DNA, Viral/genetics , Drug Resistance, Viral/genetics , Genotype , Hepatitis B virus/genetics , Humans , Mutation , SARS-CoV-2/geneticsABSTRACT
The pandemic of coronavirus disease 2019 (COVID-19), due to the novel coronavirus (SARS-CoV-2), has created an unprecedented threat to the global health system, especially in resource-limited areas. This challenge shines a spotlight on the urgent need for a point-of-care (POC) quantitative real-time PCR (qPCR) test for sensitive and rapid diagnosis of viral infections. In a POC system, a closed, single-use, microfluidic cartridge is commonly utilized for integration of nucleic acid preparation, PCR amplification and florescence detection. But, most current cartridge systems often involve complicated nucleic acid extraction via active pumping that relies on cumbersome external hardware, causing increases in system complexity and cost. In this work, we demonstrate a gravity-driven cartridge design for an integrated viral RNA/DNA diagnostic test that does not require auxiliary hardware for fluid pumping due to adopted extraction-free amplification. This microfluidic cartridge only contains two reaction chambers for nucleic acid lysis and amplification respectively, enabling a fast qPCR test in less than 30 min. This gravity-driven pumping strategy can help simplify and minimize the microfluidic cartridge, thus enabling high-throughput (up to 12 test cartridges per test) molecular detection via a small cartridge readout system. Thus, this work addresses the scalability limitation of POC molecular testing and can be run in any settings. We verified the analytical sensitivity and specificity of the cartridge testing for respiratory pathogens and sexually transmitted diseases using SARS-CoV-2, influenza A/B RNA samples, and human papillomavirus 16/18 DNA samples. Our cartridge system exhibited a comparable detection performance to the current gold standard qPCR instrument ABI 7500. Moreover, our system showed very high diagnostic accuracy for viral RNA/DNA detection that was well validated by ROC curve analysis. The sample-to-answer molecular testing system reported in this work has the advantages of simplicity, rapidity, and low cost, making it highly promising for prevention and control of infectious diseases in poor-resource areas.
Subject(s)
COVID-19 , Influenza, Human , COVID-19/diagnosis , COVID-19 Testing , DNA, Viral/genetics , Human papillomavirus 16/genetics , Humans , Influenza, Human/diagnosis , Microfluidics , Nucleic Acid Amplification Techniques , RNA, Viral/analysis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and SpecificityABSTRACT
BACKGROUND: Chronic hepatitis B virus (HBV) treatment consists of nucleos(t)ide analogues to suppress viral replication. The HBV inhibitor tenofovir has a high barrier to resistance, however, evidence of virus-escape is emerging. This study investigates HBV evolution in patients undergoing tenofovir treatment with the primary aim to assess the emergence of putative resistance mutations. METHODS: HBV DNA was extracted from blood samples of two patients with HBeAg-positive chronic HBV infection and persistent viremia despite tenofovir treatment, and subsequently amplified by PCR before full-length HBV genomes were assembled by deep sequencing. The mutation linkage within the viral population was evaluated by clonal analysis of amplicons. RESULTS: Sequence analysis of HBV, derived from 11 samples collected 2010-2020 from one patient, identified 12 non-synonymous single-nucleotide polymorphisms (SNPs) emerging during a tenofovir treatment interruption from 2014 to 2017. Two of the SNPs were in the reverse transcriptase (RT; H35Q and D263E). The two RT mutations were linked and persisted despite restarting tenofovir treatment in 2017. For the second patient, we analyzed HBV derived from six samples collected 2014-2020 following 10 years of tenofovir treatment, and identified five non-synonymous SNPs, that confer resistance towards entecavir and/or lamivudine. Two RT mutations (H35N and P237T) emerged during subsequent 5-year entecavir treatment. H35N was maintained during final tenofovir treatment. CONCLUSIONS: Our findings indicate that changes at the conserved residue 35 (H35N/Q) in the HBV RT may be associated with tenofovir resistance. These variants have not previously been described, and further studies are warranted to assess resistance in vitro and in vivo.
Subject(s)
Hepatitis B, Chronic , Organophosphonates , Adenine/adverse effects , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , DNA, Viral/genetics , Drug Resistance, Viral/genetics , Hepatitis B virus/genetics , Hepatitis B, Chronic/drug therapy , Humans , Mutation , Organophosphonates/therapeutic use , RNA-Directed DNA Polymerase/genetics , Tenofovir/pharmacology , Tenofovir/therapeutic use , Viremia/drug therapyABSTRACT
BACKGROUND: Torquetenovirus (TTV), a widespread anellovirus recognized as the main component of the healthy human virome, displays viremia that is highly susceptible to variations in immune competence. TTV possesses microRNA (miRNA)-coding sequences that might be involved in viral immune evasion. Among TTV-encoded miRNAs, miRNA t1a, t3b, and tth8 have been found in biological fluids. Here, the presence of TTV DNA and TTV miRNAs in the plasma of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected subjects was investigated to monitor the possible association with coronavirus disease 2019 (COVID-19) severity. METHODS: Detection of TTV DNA and miRNA t1a, t3b, and tth8 was investigated in plasma samples of 56 SARS-CoV-2-infected subjects with a spectrum of different COVID-19 outcomes. TTV DNA and TTV miRNAs were assessed with a universal single step real-time TaqMan PCR assay and miRNA quantitative RT-PCR miRNA assay, respectively. RESULTS: The TTV DNA prevalence was 59%, whereas at least one TTV miRNA was found in 94% of the patients tested. miRNA tth8 was detected in 91% of subjects, followed by miRNAs t3b (64%) and miRNAt1a (30%). Remarkably, although TTV DNA was unrelated to COVID-19 severity, miRNA tth8 was significantly associated with the degree of disease (adjusted incidence rate ratio (IRR) 2.04, 95% CI 1.14-3.63, for the subjects in the high severity group compared to those in the low severity group). CONCLUSIONS: Our findings encourage further investigation to understand the potential role of TTV miRNAs in the different outcomes of COVID-19 at early and late stages.
Subject(s)
COVID-19 , MicroRNAs , Torque teno virus , DNA, Viral/genetics , Humans , MicroRNAs/genetics , SARS-CoV-2/genetics , Torque teno virus/geneticsABSTRACT
Adenoviruses (AdVs) are widespread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver, and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID-19 vaccination. AdV vectors are physically and genetically stable and generally safe in humans. The particles have an icosahedral coat and a nucleoprotein core with a DNA genome. We describe the concept of AdV cell entry and highlight recent advances in cytoplasmic transport, uncoating, and nuclear import of the viral DNA. We highlight a recently discovered "linchpin" function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3 ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover mechanisms controlling capsid stability and premature uncoating and provide insight into nuclear transport of nucleic acids.
Subject(s)
Adenoviridae , COVID-19 , Animals , Humans , Active Transport, Cell Nucleus , Adenoviridae/genetics , Adenoviridae/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Proteasome Endopeptidase Complex/metabolism , Kinesins , COVID-19 Vaccines , Nuclear Pore/genetics , Nuclear Pore/metabolism , Capsid Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , Nucleoproteins/metabolism , Mammals/genetics , Mammals/metabolismABSTRACT
INTRODUCTION: This study investigated the spontaneous clinical course of patients with endomyocardial biopsy (EMB)-proven lymphocytic myocarditis and cardiac human herpesvirus 6 (HHV6) DNA presence, and the effectiveness of steroid-based intervention in HHV6-positive patients. RESULTS: 756 heart failure (HF) patients underwent an EMB procedure to determine the underlying cause of unexplained HF. Low levels of HHV6 DNA, detectable by nested PCR only, were found in 10.4% of the cases (n = 79) of which 62% (n = 49) showed myocardial inflammation. The spontaneous course of patients with EMB-proven HHV6 DNA-associated lymphocytic myocarditis (n = 26) showed significant improvements in the left ventricular ejection fraction (LVEF) and clinical symptoms, respectively, in 15/26 (60%) patients, 3-12 months after disease onset. EMB mRNA expression of components of the NLRP3 inflammasome pathway and protein analysis of cardiac remodeling markers, analyzed by real-time PCR and MALDI mass spectrometry, respectively, did not differ between HHV6-positive and -negative patients. In another cohort of patients with ongoing symptoms related to lymphocytic myocarditis associated with cardiac levels of HHV6-DNA copy numbers <500 copies/µg cardiac DNA, quantified by real-time PCR, the efficacy and safety of steroid-based immunosuppression for six months was investigated. Steroid-based immunosuppression improved the LVEF (≥5%) in 8/10 patients and reduced cardiac inflammation in 7/10 patients, without an increase in cardiac HHV6 DNA levels in follow-up EMBs. CONCLUSION: Low HHV6 DNA levels are frequently detected in the myocardium, independent of inflammation. In patients with lymphocytic myocarditis with low levels of HHV6 DNA, the spontaneous clinical improvement is nearby 60%. In selected symptomatic patients with cardiac HHV6 DNA copy numbers less than 500 copies/µg cardiac DNA and without signs of an active systemic HHV6 infection, steroid-based therapy was found to be effective and safe. This finding needs to be further confirmed in large, randomized trials.
Subject(s)
Herpesvirus 6, Human/physiology , Immunosuppressive Agents/administration & dosage , Myocarditis/drug therapy , Myocarditis/virology , Roseolovirus Infections/drug therapy , Roseolovirus Infections/virology , Steroids/administration & dosage , Adult , Aged , Biopsy , Cohort Studies , DNA, Viral/genetics , Female , Gene Dosage , Herpesvirus 6, Human/genetics , Herpesvirus 6, Human/isolation & purification , Humans , Male , Middle Aged , Myocarditis/immunology , Myocarditis/physiopathology , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Roseolovirus Infections/immunology , Roseolovirus Infections/physiopathology , Stroke VolumeABSTRACT
A 67-year-old male veterinarian presented with fatigue, anorexia, and diarrhea. Although there were no tick bite marks, we suspected severe fever with thrombocytopenia syndrome (SFTS) due to bicytopenia, mild disturbance of consciousness, and a history of outdoor activities. Thus, we started immunoglobulin therapy immediately. A serum reverse transcription-polymerase chain reaction (RT-PCR) test for SFTS virus (SFTSV) was positive. The patient had treated a cat with thrombocytopenia 10 days prior to admission. The cat's serum SFTSV RT-PCR test result was positive, and the whole genome sequences of the patient's and cat's SFTSV were identical, suggesting the possibility of transmission from the cat to the patient. Other cases of direct cat-to-human SFTV transmission have been reported recently. Mucous membranes should be protected, including eye protection, in addition to standard precautions, when in contact with any cat with suspected SFTS.
Subject(s)
Cat Diseases/virology , Severe Fever with Thrombocytopenia Syndrome/transmission , Severe Fever with Thrombocytopenia Syndrome/virology , Aged , Animals , Cat Diseases/blood , Cats , DNA, Viral/blood , DNA, Viral/genetics , Humans , Male , Phlebovirus/classification , Phlebovirus/genetics , Phlebovirus/isolation & purification , Severe Fever with Thrombocytopenia Syndrome/blood , Severe Fever with Thrombocytopenia Syndrome/diagnosis , VeterinariansABSTRACT
Interferon gamma (IFN-γ) is a crucial cytokine in host immune response to hepatitis B virus (HBV) infection. This study aimed to determine whether a functional polymorphism +874T/A in IFN-γ gene linked to high and low producer phenotypes [IFN-γ (+874Thigh â Alow)] may alter the outcomes of chronic HBV infection in Tunisian population. The +874T/A was analysed by ARMS-PCR method in the group of 200 patients chronically infected with HBV and 200 healthy controls. We observed that minor +874A allele, minor +874AA and +874TA genotypes were significantly more frequent in the chronic hepatitis B group in comparison to the control group [49 vs. 31%, P < 10-4; 24 vs. 13%, P < 10-4; 52 vs. 38%, P < 10-4; respectively]. Besides, they were associated with susceptibility to hepatitis B infection [OR = 2.15, 3.87 and 2.84, respectively]. The minor +874A allele and +874AA genotype were statistically more representative in the sub-group of patients with high viral DNA load when compared with the sub-group of patients with low HBV DNA load [(57% vs. 43%, P = 0.003, OR = 1.79); (33% vs. 14%, P = 0.003, OR = 3.59), respectively]. Collectively, our study suggests an association between the IFN-γ +874T/A SNP and persistence of HBV by the enhancement of HBV DNA replication.
Subject(s)
DNA Replication , Genetic Association Studies , Genetic Predisposition to Disease , Hepatitis B virus/physiology , Hepatitis B, Chronic/genetics , Interferon-gamma/genetics , Polymorphism, Single Nucleotide/genetics , Virus Replication/physiology , Adult , Alleles , Case-Control Studies , DNA, Viral/genetics , Female , Gene Frequency/genetics , Hepatitis B, Chronic/virology , Humans , Male , Viral Load/geneticsABSTRACT
BACKGROUND: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA. METHODS: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels. RESULTS: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. CONCLUSIONS: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.
Subject(s)
Bacteriophage T4/enzymology , DNA, Viral/chemistry , DNA-Binding Proteins/chemistry , Membrane Proteins/chemistry , Nucleic Acid Amplification Techniques , SARS-CoV-2/chemistry , Viral Proteins/chemistry , DNA, Viral/genetics , SARS-CoV-2/geneticsABSTRACT
Quantitative physicochemical perspective on life processes has been a great asset, in bioengineering and biotechnology. The quantitative physicochemical approach can be applied to practically all organisms, including viruses, if their chemical composition and thermodynamic properties are known. In this paper, a new method is suggested for determining elemental composition of viruses, based on atom counting. The atom counting method requires knowledge of genetic sequence, protein sequences and protein copy numbers. An algorithm was suggested for a program that finds elemental composition of various viruses (DNA or RNA, enveloped or non-enveloped). Except for the nucleic acid, capsid proteins, lipid bilayer and carbohydrates, this method includes membrane proteins, as well as spike proteins. The atom counting method has been compared with the existing molecular composition and geometric methods on 5 viruses of different morphology, as well as experimentally determined composition of the poliovirus. The atom counting method was found to be more accurate in most cases. The three methods were found to be complementary, since they require different kind of input information. Moreover, since the 3 methods rest on different assumptions, results of one model can be compared to those of the other two.
Subject(s)
Viruses/chemistry , Algorithms , Animals , Carbohydrates/chemistry , Chemical Phenomena , Computational Biology , DNA, Viral/chemistry , DNA, Viral/genetics , Elements , Environmental Science , Humans , Lipids/chemistry , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Thermodynamics , Viral Proteins/chemistry , Viral Proteins/genetics , Viruses/geneticsSubject(s)
Acute-On-Chronic Liver Failure/complications , Acute-On-Chronic Liver Failure/mortality , COVID-19/complications , Hepatitis B virus/genetics , Hepatitis B, Chronic/complications , Liver Cirrhosis/complications , SARS-CoV-2/isolation & purification , Adult , Antiviral Agents/therapeutic use , COVID-19/diagnosis , COVID-19/virology , DNA, Viral/genetics , Fatal Outcome , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/virology , Humans , Male , Tenofovir/therapeutic use , Treatment OutcomeABSTRACT
The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.
Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , CRISPR-Cas Systems , DNA, Viral/genetics , Mutation , SARS-CoV-2/genetics , Biosensing Techniques , COVID-19/virology , DNA/analysis , Genetic Techniques , HEK293 Cells , Humans , Immunoglobulin G/chemistry , Peptide Library , Polymers/chemistry , Spain/epidemiology , Surface Plasmon ResonanceABSTRACT
Bats are an important reservoir of several zoonotic diseases. However, the circulation of bat coronaviruses (BatCoV) in live animal markets in Indonesia has not been reported. Genetic characterization of BatCoV was performed by sequencing partial RdRp genes. Real-time polymerase chain reaction based on nucleocapsid protein (N) gene and Enzyme-linked immunosorbent assay against the N protein were conducted to detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA and antibody, respectively. We identified the presence of BatCoV on Cynopterus brachyotis, Macroglossus minimus, and Rousettus amplexicaudatus. The results showed that the BatCoV included in this study are from an unclassified coronavirus group. Notably, SARS-CoV-2 viral RNA and antibodies were not detected in the sampled bats.
Subject(s)
Chiroptera/virology , Coronavirus/classification , Coronavirus/isolation & purification , Animals , Coronavirus/genetics , DNA, Viral/genetics , Enzyme-Linked Immunosorbent Assay/veterinary , Indonesia , Nucleocapsid Proteins/genetics , Real-Time Polymerase Chain Reaction/veterinary , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Species SpecificityABSTRACT
Cassava plantations in an area of 458 hectares spanning five provinces along the Thailand-Cambodia border were surveyed from October 2018 to July 2019 to determine the prevalence of cassava mosaic disease (CMD) caused by Sri Lankan cassava mosaic virus (SLCMV) in the region. CMD prevalence was 40% in the whole area and 80% in Prachinburi, 43% in Sakaeo, 37% in Burium, 25% in Surin, and 19% in Sisaket provinces. Disease incidence of CMD was highest 43.08% in Sakaeo, followed by 26.78% in Prachinburi, 7% in Burium, 2.58% in Surin, and 1.25% in Sisaket provinces. Disease severity of CMD symptoms was mild chlorosis to moderate mosaic (2-3). The greatest disease severity was recorded in Prachinburi and Sakaeo provinces. Asymptomatic plants were identified in Surin (12%), Prachinburi (5%), Sakaeo (0.2%), and Buriram (0.1%) by PCR analysis. Cassava cultivars CMR-89 and Huai Bong 80 were susceptible to CMD. In 95% of cases, the infection was transmitted by whiteflies (Bemisia tabaci), which were abundant in Sakaeo, Buriram, and Prachinburi but were sparse in Surin; their densities were highest in May and June 2019. Nucleotide sequencing of the mitochondrial cytochrome oxidase 1 (mtCO1) gene of whiteflies in Thailand revealed that it was similar to the mtCO1 gene of Asia II 1 whitefly. Furthermore, the AV1 gene of SLCMV-which encodes the capsid protein-showed 90% nucleotide identity with SLCMV. Phylogenetic analysis of completed nucleotide sequences of DNA-A and DNA-B components of the SLCMV genome determined by rolling circle amplification (RCA) indicated that they were similar to the nucleotide sequence of SLCMV isolates from Thailand, Vietnam, and Cambodia. These results provide important insights into the distribution, impact, and spread of CMD and SLCMV in Thailand.
Subject(s)
Begomovirus/genetics , Animals , Base Sequence/genetics , Cambodia , DNA, Viral/genetics , Hemiptera/virology , Plant Diseases/virology , Plants/virology , Thailand , VietnamABSTRACT
The World Health Organization global call to eliminate cervical cancer encourages countries to consider introducing or improving cervical cancer screening programs. Brazil's Unified Health System (SUS) is among the world's largest public health systems offering free cytology testing, follow-up colposcopy, and treatment. Yet, health care networks across the country have unequal infrastructure, human resources, equipment, and supplies resulting in uneven program performance and large disparities in cervical cancer incidence and mortality. An effective screening program needs multiple strategies feasible for each community's reality, facilitating coverage and follow-up adherence. Prioritizing those at highest risk with tests that better stratify risk will limit inefficiencies, improving program impact across different resource settings. Highly sensitive human papillomavirus (HPV)-DNA testing performs better than cytology and, with self-collection closer to homes and workplaces, improves access, even in remote regions. Molecular triage strategies like HPV genotyping can identify from the same self-collected sample, those at highest risk requiring follow-up. If proven acceptable, affordable, cost-effective, and efficient in the Brazilian context, these strategies would increase coverage while removing the need for speculum exams for routine screening and reducing follow-up visits. SUS could implement a nationwide organized program that accommodates heterogenous settings across Brazil, informing a variety of screening programs worldwide.
Subject(s)
COVID-19/complications , Cytodiagnosis/methods , Early Detection of Cancer/methods , Papillomaviridae/isolation & purification , Papillomavirus Infections/complications , SARS-CoV-2/isolation & purification , Uterine Cervical Neoplasms/diagnosis , Brazil/epidemiology , DNA, Viral/analysis , DNA, Viral/genetics , Female , Humans , Papillomavirus Infections/genetics , Papillomavirus Infections/virology , Uterine Cervical Neoplasms/epidemiology , Uterine Cervical Neoplasms/virologyABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics. IMPORTANCE To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public.
Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , DNA Contamination , DNA, Viral/genetics , SARS-CoV-2/genetics , False Positive Reactions , Humans , Molecular Diagnostic Techniques , RNA, Viral/genetics , SARS-CoV-2/isolation & purificationABSTRACT
BACKGROUND: Pathogen reduction technologies (PRT) based on nucleic-acid damaging chemicals and/or irradiation are increasingly being used to increase safety of blood components against emerging pathogens, such as convalescent plasma in the ongoing COVID-19 pandemic. Current methods for PRT validation are limited by the resources available to the blood component manufacturer, and quality control rely over pathogen spiking and hence invariably require sacrifice of the tested blood units: quantitative real-time PCR is the current pathogen detection method but, due to the high likelihood of detecting nonviable fragments, requires downstream pathogen culture. We propose here a new molecular validation of PRT based on the highly prevalent human symbiont torquetenovirus (TTV) and rolling circle amplification (RCA). MATERIALS AND METHODS: Serial apheresis plasma donations were tested for TTV before and after inactivation with Intercept® PRT using real-time quantitative PCR (conventional validation), RCA followed by real-time PCR (our validation), and reverse PCR (for cross-validation). RESULTS: While only 20% of inactivated units showed significant decrease in TTV viral load using real-time qPCR, all donations tested with RCA followed by real-time PCR showed TTV reductions. As further validation, 2 units were additionally tested with reverse PCR, which confirmed absence of entire circular genomes. DISCUSSION: We have described and validated a conservative and easy-to-setup protocol for molecular validation of PRT based on RCA and real-time PCR for TTV.