The source of the severe acute respiratory syndrome (SARS) epidemic was traced to wildlife market civets and ultimately to bats. Subsequent hunting for novel coronaviruses (CoVs) led to the discovery ...of two additional human and over 40 animal CoVs, including the prototype lineage C betacoronaviruses, Tylonycteris bat CoV HKU4 and Pipistrellus bat CoV HKU5; these are phylogenetically closely related to the Middle East respiratory syndrome (MERS) CoV, which has affected more than 1,000 patients with over 35% fatality since its emergence in 2012. All primary cases of MERS are epidemiologically linked to the Middle East. Some of these patients had contacted camels which shed virus and/or had positive serology. Most secondary cases are related to health care-associated clusters. The disease is especially severe in elderly men with comorbidities. Clinical severity may be related to MERS-CoV's ability to infect a broad range of cells with DPP4 expression, evade the host innate immune response, and induce cytokine dysregulation. Reverse transcription-PCR on respiratory and/or extrapulmonary specimens rapidly establishes diagnosis. Supportive treatment with extracorporeal membrane oxygenation and dialysis is often required in patients with organ failure. Antivirals with potent in vitro activities include neutralizing monoclonal antibodies, antiviral peptides, interferons, mycophenolic acid, and lopinavir. They should be evaluated in suitable animal models before clinical trials. Developing an effective camel MERS-CoV vaccine and implementing appropriate infection control measures may control the continuing epidemic.
In late 2019, a novel human coronavirus – severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) – emerged in Wuhan, China. This virus has caused a global pandemic involving more than 200 ...countries. SARS-CoV-2 is highly adapted to humans and readily transmits from person-to-person.
To investigate the infectivity of SARS-CoV-2 under various environmental and pH conditions. The efficacies of various laboratory virus inactivation methods and home disinfectants against SARS-CoV-2 were investigated.
The residual virus in dried form or in solution was titrated on to Vero E6 cells on days 0, 1, 3, 5 and 7 after incubation at different temperatures. Viral viability was determined after treatment with various disinfectants and pH solutions at room temperature (20–25oC).
SARS-CoV-2 was able to retain viability for 3–5 days in dried form or 7 days in solution at room temperature. SARS-CoV-2 could be detected under a wide range of pH conditions from pH 4 to pH 11 for several days, and for 1–2 days in stool at room temperature but lost 5 logs of infectivity. A variety of commonly used disinfectants and laboratory inactivation procedures were found to reduce viral viability effectively.
This study demonstrated the stability of SARS-CoV-2 on environmental surfaces, and raises the possibility of faecal–oral transmission. Commonly used fixatives, nucleic acid extraction methods and heat inactivation were found to reduce viral infectivity significantly, which could ensure hospital and laboratory safety during the SARS-CoV-2 pandemic.
Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation ...profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.
Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome that elevates the risk of hepatocellular carcinoma (HCC). Although alteration of lipid metabolism has ...been increasingly recognized as a hallmark of cancer cells, the deregulated metabolic modulation of HCC cells in the NAFLD progression remains obscure. Here, we discovers an endoplasmic reticulum-residential protein, Nogo-B, as a highly expressed metabolic modulator in both murine and human NAFLD-associated HCCs, which accelerates high-fat, high-carbohydrate diet-induced metabolic dysfunction and tumorigenicity. Mechanistically, CD36-mediated oxLDL uptake triggers CEBPβ expression to directly upregulate Nogo-B, which interacts with ATG5 to promote lipophagy leading to lysophosphatidic acid-enhanced YAP oncogenic activity. This CD36-Nogo-B-YAP pathway consequently reprograms oxLDL metabolism and induces carcinogenetic signaling for NAFLD-associated HCCs. Targeting the Nogo-B pathway may represent a therapeutic strategy for HCC arising from the metabolic syndrome.
Automated point-of-care molecular assays have greatly shortened the turnaround time of respiratory virus testing. One of the major bottlenecks now lies at the specimen collection step, especially in ...a busy clinical setting. Saliva is a convenient specimen type that can be provided easily by adult patients. This study assessed the diagnostic validity, specimen collection time and cost associated with the use of saliva.
This was a prospective diagnostic validity study comparing the detection rate of respiratory viruses between saliva and nasopharyngeal aspirate (NPA) among adult hospitalized patients using Xpert® Xpress Flu/RSV. The cost and time associated with the collection of saliva and nasopharyngeal specimens were also estimated.
Between July and October 2017, 214 patients were recruited. The overall agreement between saliva and NPA was 93.3% (196/210, κ 0.851, 95% CI 0.776–0.926). There was no significant difference in the detection rate of respiratory viruses between saliva and NPA (32.9% (69/210) versus 35.7% (75/210); p 0.146). The overall sensitivity and specificity were 90.8% (81.9%–96.2%) and 100% (97.3%–100%), respectively, for saliva, and were 96.1% (88.9%–99.2%) and 98.5% (94.7%–99.8%), respectively, for NPA. The time and cost associated with the collection of saliva were 2.26-fold and 2.59-fold lower, respectively, than those of NPA.
Saliva specimens have high sensitivity and specificity in the detection of respiratory viruses by an automated multiplex Clinical Laboratory Improvement Amendments-waived point-of-care molecular assay when compared with those of NPA. The use of saliva also reduces the time and cost associated with specimen collection.
Cell-free DNA (cfDNA) in human plasma is a class of biomarkers with many current and potential future diagnostic applications. Recent studies have shown that cfDNA molecules are not randomly ...fragmented and possess information related to their tissues of origin. Pathologies causing death of cells from particular tissues result in perturbations in the relative distribution of DNA from the affected tissues. Such tissue-of-origin analysis is particularly useful in the development of liquid biopsies for cancer. It is therefore of value to accurately determine the relative contributions of the tissues to the plasma DNA pool in a simultaneous manner. In this work, we report that in open chromatin regions, cfDNA molecules show characteristic fragmentation patterns reflected by sequencing coverage imbalance and differentially phased fragment end signals. The latter refers to differences in the read densities of sequences corresponding to the orientation of the upstream and downstream ends of cfDNA molecules in relation to the reference genome. Such cfDNA fragmentation patterns preferentially occur in tissue-specific open chromatin regions where the corresponding tissues contributed DNA into the plasma. Quantitative analyses of such signals allow measurement of the relative contributions of various tissues toward the plasma DNA pool. These findings were validated by plasma DNA sequencing data obtained from pregnant women, organ transplantation recipients, and cancer patients. Orientation-aware plasma DNA fragmentation analysis therefore has potential diagnostic applications in noninvasive prenatal testing, organ transplantation monitoring, and cancer liquid biopsy.
Abstract
Background
Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and ...cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends.
Methods
Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing.
Results
The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83.
Conclusions
Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.
Abstract
Background
A novel coronavirus of zoonotic origin (2019-nCoV) has recently been identified in patients with acute respiratory disease. This virus is genetically similar to SARS coronavirus ...and bat SARS-like coronaviruses. The outbreak was initially detected in Wuhan, a major city of China, but has subsequently been detected in other provinces of China. Travel-associated cases have also been reported in a few other countries. Outbreaks in health care workers indicate human-to-human transmission. Molecular tests for rapid detection of this virus are urgently needed for early identification of infected patients.
Methods
We developed two 1-step quantitative real-time reverse-transcription PCR assays to detect two different regions (ORF1b and N) of the viral genome. The primer and probe sets were designed to react with this novel coronavirus and its closely related viruses, such as SARS coronavirus. These assays were evaluated using a panel of positive and negative controls. In addition, respiratory specimens from two 2019-nCoV-infected patients were tested.
Results
Using RNA extracted from cells infected by SARS coronavirus as a positive control, these assays were shown to have a dynamic range of at least seven orders of magnitude (2x10−4-2000 TCID50/reaction). Using DNA plasmids as positive standards, the detection limits of these assays were found to be below 10 copies per reaction. All negative control samples were negative in the assays. Samples from two 2019-nCoV-infected patients were positive in the tests.
Conclusions
The established assays can achieve a rapid detection of 2019n-CoV in human samples, thereby allowing early identification of patients.
Circulating tumor-derived DNA testing for cancer screening has recently been demonstrated in a prospective study on identification of nasopharyngeal carcinoma (NPC) among 20,174 asymptomatic ...individuals. Plasma EBV DNA, a marker for NPC, was detected using real-time PCR. While plasma EBV DNA was persistently detectable in 97.1% of the NPCs identified, ∼5% of the general population had transiently detectable plasma EBV DNA. We hypothesized that EBV DNA in plasma of subjects with or without NPC may have different molecular characteristics. We performed target-capture sequencing of plasma EBV DNA and identified differences in the abundance and size profiles of EBV DNA molecules within plasma of NPC and non-NPC subjects. NPC patients had significantly higher amounts of plasma EBV DNA, which showed longer fragment lengths. Cutoff values were established from an exploratory dataset and tested in a validation sample set. Adopting an algorithm that required a sample to concurrently pass cutoffs for EBV DNA counting and size measurements, NPCs were detected at a positive predictive value (PPV) of 19.6%. This represented superior performance compared with the PPV of 11.0% in the prospective screening study, which required participants with an initially detectable plasma EBV DNA result to be retested within 4 weeks. The observed differences in the molecular nature of EBV DNA molecules in plasma of subjects with or without NPC were successfully translated into a sequencing-based test that had a high PPV for NPC screening and achievable through single time-point testing.
Cell-free DNA in human plasma is nonrandomly fragmented and reflects genomewide nucleosomal organization. Previous studies had demonstrated tissue-specific preferred end sites in plasma DNA of ...pregnant women. In this study, we performed integrative analysis of preferred end sites with the size characteristics of plasma DNA fragments. We mined the preferred end sites in short and long plasma DNA molecules separately and found that these “size-tagged” ends showed improved accuracy in fetal DNA fraction estimation and enhanced noninvasive fetal trisomy 21 testing. Further analysis revealed that the fetal and maternal preferred ends were generated from different locations within the nucleosomal structure. Hence, fetal DNA was frequently cut within the nucleosome core while maternal DNA was mostly cut within the linker region. We further demonstrated that the nucleosome accessibility in placental cells was higher than that for white blood cells, which might explain the difference in the cutting positions and the shortness of fetal DNA in maternal plasma. Interestingly, short and long size-tagged ends were also observable in the plasma of nonpregnant healthy subjects and demonstrated size differences similar to those in the pregnant samples. Because the nonpregnant samples did not contain fetal DNA, the data suggested that the interrelationship of preferred DNA ends, chromatin accessibility, and plasma DNA size profile is likely a general one, extending beyond the context of pregnancy. Plasma DNA fragment end patterns have thus shed light on production mechanisms and show utility in future developments in plasma DNA-based noninvasive molecular diagnostics.
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