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1.
PLoS One ; 15(9): e0236311, 2020.
Article in English | MEDLINE | ID: covidwho-748965

ABSTRACT

Since SARS-CoV-2-based disease (COVID-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription PCR (rtPCR) results, raises as a major clinical need. Here we evaluated the performance of a ddPCR-based assay to quantify SARS-CoV-2 titer in 55 suspected COVID-19 cases with negative rtPCR results thanks to in-house ddPCR assay (targeting RdRp and host RNaseP). Samples were collected at ASST-GOM Niguarda between February and May 2020 at hospital admission. Clinical and imaging data were obtained for clinical staging and definition of disease severity. Patients were mainly female (45.5%) with a median age of 73 (57-84) years. ddPCR-based assay detected SARS-CoV-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/mL, IQR: 72-345). In 15 of them (78.9%), chest CT showed a classical COVID-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe COVID-19 manifestations. ddPCR did not identify any trace of SARS-CoV-2 genome in the respiratory samples of the remaining 36 patients. The serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of SARS-CoV-2 antibodies in all patients tested positive for SARS-CoV-2 in ddPCR (100%). Contrariwise, negative tests were observed in 95.0% ddPCR negative patients (P<0.001). Thanks to a ddPCR-based assay, we achieved a rapid and accurate SARS-CoV-2 diagnosis in rtPCR-negative respiratory samples of individuals with COVID-19 suspect, allowing the rapid taking care and correct management of these patients.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Nasopharynx/virology , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Aged , Aged, 80 and over , Betacoronavirus/isolation & purification , Coronavirus Infections/pathology , Coronavirus Infections/virology , Female , Humans , Limit of Detection , Male , Middle Aged , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , RNA, Viral/metabolism , Severity of Illness Index , Viral Load
2.
PLoS One ; 15(8): e0237559, 2020.
Article in English | MEDLINE | ID: covidwho-709369

ABSTRACT

BACKGROUND: The world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV-2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5' and 3' untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon. METHODS: To have an idea of the global changes in 5' and 3'-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV-2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by 'RBPmap' and 'RNA22 v2' respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue. RESULTS: We identified 28 unique variants in SARS-CoV-2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5'-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3'UTR among most of the continents. Furthermore, we found that despite the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs. CONCLUSION: Our results, shows for the first time in SARS-Cov-2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.


Subject(s)
3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Betacoronavirus/genetics , Coronavirus Infections/metabolism , Genome, Viral/genetics , Genomic Instability/genetics , Host-Pathogen Interactions/genetics , MicroRNAs/metabolism , Pneumonia, Viral/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Base Sequence , Binding Sites , Coronavirus Infections/virology , Humans , Open Reading Frames/genetics , Pandemics , Pneumonia, Viral/virology , Protein Binding/genetics
3.
J Korean Med Sci ; 35(31): e287, 2020 Aug 10.
Article in English | MEDLINE | ID: covidwho-705844

ABSTRACT

BACKGROUND: This study was performed to compare the viral load and kinetics of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in saliva with those in standard nasopharyngeal/oropharyngeal (NP/OP) swabs. METHODS: Fifteen patients with SARS-CoV-2 infection from four hospitals were prospectively enrolled and matched samples of nasopharyngeal/oropharyngeal swabs and saliva were collected at Day 1 of admission and every other day till consequently negative for two times. Real-time reverse transcription polymerase chain reaction (rRT-PCR) was performed to detect the envelope (E) and RNA-dependent RNA polymerase (RdRP) genes. RESULTS: The cycle threshold values of saliva were comparable to those of NP/OP swabs overall (P = 0.720, Mann-Whitney U test). However, the overall sensitivity of rRT-PCR using saliva was 64% (34/53), which is lower than the 77% (41/53) using NP/OP swabs. The sensitivity of rRT-PCR using saliva was especially lower in early stage of symptom onset (1-5 days; 8/15; 53%) and in patients who did not have sputum (12/22; 55%). CONCLUSION: Saliva sample itself is not appropriate for initial diagnosis of coronavirus disease 2019 (COVID-19) to replace NP/OP swabs, especially for the person who does not produce sputum. COVID-19 cannot be excluded when the test using saliva is negative, and it is necessary to retest using NP/OP swabs.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Saliva/virology , Adolescent , Adult , Aged , Aged, 80 and over , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , Prospective Studies , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , Republic of Korea , Viral Load , Young Adult
4.
J Korean Med Sci ; 35(31): e288, 2020 Aug 10.
Article in English | MEDLINE | ID: covidwho-704430

ABSTRACT

BACKGROUND: In February 2020, a coronavirus disease 2019 (COVID-19) outbreak was reported in fitness centers in Cheonan, Korea. METHODS: From February 24 to March 13, an epidemiological investigation was conducted on the fitness center outbreak. All those who were screened were tested for severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) using real-time reverse transcriptase polymerase chain reaction. Contacts were traced and self-isolated for 14 days. We determined the epidemiological characteristics of confirmed cases of SARS-CoV-2 infection, and estimated the time-dependent reproduction number to assess the transmission dynamics of the infection. RESULTS: A total of 116 cases were confirmed, and 1,687 contacts were traced. The source cases were 8 Zumba instructors who led aerobics classes in 10 fitness centers, and had the largest average number of contacts. A total of 57 Zumba class participants, 37 of their family members, and 14 other contacts were confirmed as cases. The attack rate was 7.3%. The contacts at Zumba classes and homes had a higher attack rate than other contacts. The mean serial interval (± standard deviation) were estimated to be 5.2 (± 3.8) days. The time-dependent reproduction number was estimated to be 6.1 at the beginning of the outbreak, but it dropped to less than 1, 2 days after the epidemiological investigation was launched. CONCLUSION: The results suggest that the COVID-19 outbreak was effectively contained with rigorous contact tracing, isolating, and testing in combination with social distancing without a lock-down.


Subject(s)
Coronavirus Infections/epidemiology , Pneumonia, Viral/epidemiology , Adult , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Contact Tracing , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Disease Outbreaks , Female , Fitness Centers , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Quarantine , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , Republic of Korea/epidemiology
5.
PLoS One ; 15(8): e0237127, 2020.
Article in English | MEDLINE | ID: covidwho-695633

ABSTRACT

BACKGROUND: The global pandemic of Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV2) has resulted in unprecedented challenges for healthcare systems. One barrier to widespread testing has been a paucity of traditional respiratory viral swab collection kits relative to the demand. Whether other sample collection kits, such as widely available MRSA nasal swabs can be used to detect SARS-CoV-2 is unknown. METHODS: We compared simultaneous nasal MRSA swabs (COPAN ESwabs ® 480C flocked nasal swab in 1mL of liquid Amies medium) and virals wabs (BD H192(07) flexible mini-tip flocked nasopharyngeal swabs in 3mL Universal Transport Medium) for SARS-CoV-2 PCR testing using Simplexa COVID-19 Direct assay on patients over a 4-day period. When the results were discordant, the viral swab sample was run again on the Cepheid Xpert Xpress ® SARS-CoV-2 assay. RESULTS: Of the 81 included samples, there were 19 positives and 62 negatives in viral media and 18 positives and 63 negative in the MRSA swabs. Amongst all included samples, there was concordance between the COPAN ESwabs ® 480C and the viral swabs in 78 (96.3%). CONCLUSION: We found a high rate of concordance in test results between COPAN ESwabs ® 480C in Amies solution and BD H192(07) nasopharyngeal swabs in in 3 mL of Universal Viral Transport medium viral media. Clinicians and laboratories should feel better informed and assured using COPAN ESwabs ® 480C to help in the diagnosis of COVID-19.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Methicillin-Resistant Staphylococcus aureus/genetics , Pneumonia, Viral/diagnosis , Specimen Handling/methods , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Humans , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Nasopharynx/microbiology , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , RNA Stability , RNA, Bacterial/analysis , RNA, Bacterial/metabolism , RNA, Viral/analysis , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction
6.
Int J Mol Sci ; 21(15)2020 Jul 29.
Article in English | MEDLINE | ID: covidwho-693630

ABSTRACT

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.


Subject(s)
Betacoronavirus/genetics , Colorimetry/methods , Coronavirus Infections/diagnosis , Mass Screening/economics , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Betacoronavirus/isolation & purification , Colorimetry/economics , Coronavirus Infections/virology , Humans , Limit of Detection , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/virology , Point-of-Care Systems , RNA, Viral/metabolism , Viral Load
7.
Int J Mol Sci ; 21(15)2020 Aug 04.
Article in English | MEDLINE | ID: covidwho-693534

ABSTRACT

The current COronaVIrus Disease 2019 (COVID-19) pandemic started in December 2019. COVID-19 cases are confirmed by the detection of SARS-CoV-2 RNA in biological samples by RT-qPCR. However, limited numbers of SARS-CoV-2 genomes were available when the first RT-qPCR methods were developed in January 2020 for initial in silico specificity evaluation and to verify whether the targeted loci are highly conserved. Now that more whole genome data have become available, we used the bioinformatics tool SCREENED and a total of 4755 publicly available SARS-CoV-2 genomes, downloaded at two different time points, to evaluate the specificity of 12 RT-qPCR tests (consisting of a total of 30 primers and probe sets) used for SARS-CoV-2 detection and the impact of the virus' genetic evolution on four of them. The exclusivity of these methods was also assessed using the human reference genome and 2624 closely related other respiratory viral genomes. The specificity of the assays was generally good and stable over time. An exception is the first method developed by the China Center for Disease Control and prevention (CDC), which exhibits three primer mismatches present in 358 SARS-CoV-2 genomes sequenced mainly in Europe from February 2020 onwards. The best results were obtained for the assay of Chan et al. (2020) targeting the gene coding for the spiking protein (S). This demonstrates that our user-friendly strategy can be used for a first in silico specificity evaluation of future RT-qPCR tests, as well as verifying that the former methods are still capable of detecting circulating SARS-CoV-2 variants.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Genome, Viral , Pneumonia, Viral/diagnosis , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Databases, Genetic , Humans , Open Reading Frames/genetics , Pandemics , Pneumonia, Viral/virology , Polymorphism, Single Nucleotide , RNA Replicase/genetics , RNA, Viral/analysis , Sensitivity and Specificity , Whole Genome Sequencing
8.
Eur Rev Med Pharmacol Sci ; 24(14): 7796-7800, 2020 07.
Article in English | MEDLINE | ID: covidwho-693498

ABSTRACT

The 2019 Novel Coronavirus disease (COVID-19) broke out in Wuhan, China in December 2019 and spread throughout the world. Early screening and early diagnosis play key roles in prevention and management of the epidemic. Attention should also be paid to the infection of health workers and shortage of medical resources in high-risk areas. Here, we report two cases of patients diagnosed with COVID-19 and evaluated by robotic ultrasound based on 5G-powered technology 700 km east of Wuhan. We here show the advantages of this kind of remote ultrasound scan, which could become a method for the diagnosis and assessment of COVID-19.


Subject(s)
Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Robotics , Ultrasonography/methods , Adult , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Coronavirus Infections/complications , Coronavirus Infections/virology , Female , Humans , Lung/diagnostic imaging , Male , Middle Aged , Pandemics , Pneumonia/diagnosis , Pneumonia/etiology , Pneumonia, Viral/complications , Pneumonia, Viral/virology , RNA, Viral/metabolism , Remote Sensing Technology
9.
Sensors (Basel) ; 20(15)2020 Jul 31.
Article in English | MEDLINE | ID: covidwho-693345

ABSTRACT

Coronaviruses have received global concern since 2003, when an outbreak caused by SARS-CoV emerged in China. Later on, in 2012, the Middle-East respiratory syndrome spread in Saudi Arabia, caused by MERS-CoV. Currently, the global crisis is caused by the pandemic SARS-CoV-2, which belongs to the same lineage of SARS-CoV. In response to the urgent need of diagnostic tools, several lab-based and biosensing techniques have been proposed so far. Five main areas have been individuated and discussed in terms of their strengths and weaknesses. The cell-culture detection and the microneutralization tests are still considered highly reliable methods. The genetic screening, featuring the well-established Real-time polymerase chain reaction (RT-PCR), represents the gold standard for virus detection in nasopharyngeal swabs. On the other side, immunoassays were developed, either by screening/antigen recognition of IgM/IgG or by detecting the whole virus, in blood and sera. Next, proteomic mass-spectrometry (MS)-based methodologies have also been proposed for the analysis of swab samples. Finally, virus-biosensing devices were efficiently designed. Both electrochemical immunosensors and eye-based technologies have been described, showing detection times lower than 10 min after swab introduction. Alternative to swab-based techniques, lateral flow point-of-care immunoassays are already commercially available for the analysis of blood samples. Such biosensing devices hold the advantage of being portable for on-site testing in hospitals, airports, and hotspots, virtually without any sample treatment or complicated lab precautions.


Subject(s)
Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Point-of-Care Systems , Antibodies, Viral/blood , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Betacoronavirus/metabolism , Biosensing Techniques/methods , Coronavirus Infections/virology , Humans , Immunoassay/methods , Pandemics , Pneumonia, Viral/virology , Proteomics/methods , RNA, Viral/analysis , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction/methods
10.
Sci Rep ; 10(1): 12732, 2020 07 29.
Article in English | MEDLINE | ID: covidwho-691060

ABSTRACT

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan, China in late 2019, and its resulting coronavirus disease, COVID-19, was declared a pandemic by the World Health Organization on March 11, 2020. The rapid global spread of COVID-19 represents perhaps the most significant public health emergency in a century. As the pandemic progressed, a continued paucity of evidence on routes of SARS-CoV-2 transmission has resulted in shifting infection prevention and control guidelines between classically-defined airborne and droplet precautions. During the initial isolation of 13 individuals with COVID-19 at the University of Nebraska Medical Center, we collected air and surface samples to examine viral shedding from isolated individuals. We detected viral contamination among all samples, supporting the use of airborne isolation precautions when caring for COVID-19 patients.


Subject(s)
Aerosols/analysis , Betacoronavirus/genetics , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Air Pollutants/analysis , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , Coronavirus Infections/transmission , Coronavirus Infections/virology , Humans , Infection Control/methods , Pandemics , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Public Health , RNA, Viral/isolation & purification , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
12.
Nat Commun ; 11(1): 3718, 2020 07 24.
Article in English | MEDLINE | ID: covidwho-680541

ABSTRACT

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.


Subject(s)
Methyltransferases/chemistry , RNA Caps/metabolism , Viral Nonstructural Proteins/chemistry , Viral Regulatory and Accessory Proteins/chemistry , Betacoronavirus , Coronavirus Infections/virology , Humans , Methyltransferases/metabolism , Models, Chemical , Models, Molecular , Pandemics , Pneumonia, Viral/virology , RNA, Viral/metabolism , S-Adenosylmethionine/metabolism , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , X-Ray Diffraction
13.
Nat Commun ; 11(1): 3717, 2020 07 24.
Article in English | MEDLINE | ID: covidwho-680539

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.


Subject(s)
Betacoronavirus/enzymology , Methyltransferases/chemistry , Viral Regulatory and Accessory Proteins/chemistry , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/pharmacology , Catalytic Domain , Coronavirus Infections/virology , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Methyltransferases/metabolism , Models, Chemical , Models, Molecular , Pandemics , Pneumonia, Viral/virology , RNA Caps , RNA Stability , RNA, Viral/metabolism , Viral Regulatory and Accessory Proteins/metabolism
14.
Cell ; 182(5): 1271-1283.e16, 2020 09 03.
Article in English | MEDLINE | ID: covidwho-666099

ABSTRACT

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.


Subject(s)
RNA, Messenger/genetics , RNA, Viral/genetics , Vaccines, Synthetic/immunology , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing/immunology , Binding Sites , Chlorocebus aethiops , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Female , HEK293 Cells , HeLa Cells , Humans , Immunogenicity, Vaccine , Injections, Intramuscular , Macaca fascicularis , Male , Mice , Mice, Inbred ICR , Nanoparticles/chemistry , RNA, Messenger/metabolism , RNA, Viral/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Th1 Cells/immunology , Vaccine Potency , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vero Cells , Viral Vaccines/administration & dosage , Viral Vaccines/genetics
15.
Sci Rep ; 10(1): 11887, 2020 07 17.
Article in English | MEDLINE | ID: covidwho-650165

ABSTRACT

Recently, the recurrence of positive SARS-CoV-2 viral RNA in recovered COVID-19 patients is receiving more attention. Herein we report a cohort study on the follow-up of 182 recovered patients under medical isolation observation. Twenty (10.99%) patients out of the 182 were detected to be SARS-CoV-2 RNA positive (re-positives), although none showed any clinical symptomatic recurrence, indicating that COVID-19 responds well to treatment. Patients aged under 18 years had higher re-positive rates than average, and none of the severely ill patients re-tested positive. There were no significant differences in sex between re-positives and non-re-positives. Notably, most of the re-positives turned negative in the following tests, and all of them carried antibodies against SARS-CoV-2. This indicates that they might not be infectious, although it is still important to perform regular SARS-CoV-2 RNA testing and follow-up for assessment of infectivity. The findings of this study provide information for improving the management of recovered patients, and for differentiating the follow-up of recovered patients with different risk levels.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , RNA, Viral/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Viral/blood , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Child , Child, Preschool , Cohort Studies , Coronavirus Infections/genetics , Female , Humans , Infant , Male , Middle Aged , Pandemics , Pneumonia, Viral/genetics , Recurrence , Risk , Severity of Illness Index , Young Adult
16.
Mol Biol Evol ; 37(9): 2706-2710, 2020 09 01.
Article in English | MEDLINE | ID: covidwho-641314

ABSTRACT

Due to the scope and impact of the COVID-19 pandemic there exists a strong desire to understand where the SARS-CoV-2 virus came from and how it jumped species boundaries to humans. Molecular evolutionary analyses can trace viral origins by establishing relatedness and divergence times of viruses and identifying past selective pressures. However, we must uphold rigorous standards of inference and interpretation on this topic because of the ramifications of being wrong. Here, we dispute the conclusions of Xia (2020. Extreme genomic CpG deficiency in SARS-CoV-2 and evasion of host antiviral defense. Mol Biol Evol. doi:10.1093/molbev/masa095) that dogs are a likely intermediate host of a SARS-CoV-2 ancestor. We highlight major flaws in Xia's inference process and his analysis of CpG deficiencies, and conclude that there is no direct evidence for the role of dogs as intermediate hosts. Bats and pangolins currently have the greatest support as ancestral hosts of SARS-CoV-2, with the strong caveat that sampling of wildlife species for coronaviruses has been limited.


Subject(s)
Alphacoronavirus/genetics , Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Genome, Viral , Pandemics , Pneumonia, Viral/epidemiology , Reassortant Viruses/genetics , Alphacoronavirus/classification , Alphacoronavirus/pathogenicity , Animals , Betacoronavirus/classification , Betacoronavirus/pathogenicity , Biological Evolution , Chiroptera/virology , Coronavirus Infections/immunology , Coronavirus Infections/transmission , Coronavirus Infections/virology , CpG Islands , Dogs , Eutheria/virology , Humans , Immune Evasion/genetics , Pneumonia, Viral/immunology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , RNA-Binding Proteins/metabolism , Reassortant Viruses/classification , Reassortant Viruses/pathogenicity , Virus Replication
17.
Wiley Interdiscip Rev RNA ; 11(5): e1614, 2020 09.
Article in English | MEDLINE | ID: covidwho-637124

ABSTRACT

Coronaviruses, including SARS-Cov-2, are RNA-based pathogens that interface with a large variety of RNA-related cellular processes during infection. These processes include capping, polyadenylation, localization, RNA stability, translation, and regulation by RNA binding proteins or noncoding RNA effectors. The goal of this article is to provide an in-depth perspective on the current state of knowledge of how various coronaviruses interact with, usurp, and/or avoid aspects of these cellular RNA biology machineries. A thorough understanding of how coronaviruses interact with RNA-related posttranscriptional processes in the cell should allow for new insights into aspects of viral pathogenesis as well as identify new potential avenues for the development of anti-coronaviral therapeutics. This article is categorized under: RNA in Disease and Development > RNA in Disease.


Subject(s)
Betacoronavirus/genetics , Host-Pathogen Interactions/genetics , MicroRNAs/genetics , RNA, Circular/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Animals , Betacoronavirus/metabolism , Humans , MicroRNAs/metabolism , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/metabolism , Nonsense Mediated mRNA Decay , Polyadenylation , Protein Biosynthesis , RNA Editing , RNA Splicing , RNA Stability , RNA, Circular/metabolism , RNA, Long Noncoding/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , SARS Virus/genetics , SARS Virus/metabolism
18.
Anal Chim Acta ; 1125: 57-65, 2020 Aug 15.
Article in English | MEDLINE | ID: covidwho-626172

ABSTRACT

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-coronavirus (SADS-CoV) are three emerging and re-emerging coronaviruses (CoVs). Symptoms caused by these three viruses are extremely similar, including acute diarrhea, vomiting and even death in piglets. To date, strict biosecurity is still the most effective disease prevention and control measures, and the early detection of pathogens is the most important link. Here, we developed a microfluidic-RT-LAMP chip detection system for the first time, which could detected PEDV, PDCoV and SADS-CoV simultaneously, and had advantages of rapid, simple, sensitive, high-throughput, and accurate at point-of-care settings. The lowest detection limits of the microfluidic-RT-LAMP chip method are 101 copies/µL, 102 copies/µL and 102 copies/µL for PEDV, PDCoV and SADS-CoV, respectively. The whole detection procedure can be finished rapidly in 40 min without any cross-reaction with other common swine viruses. A total of 173 clinical swine fecal samples characterized with diarrheal symptoms were used to evaluate the performance of the newly developed system, which presented good stabilities (C.V.s<5%) and specificities (100%), and possessed sensitivities of 92.24%, 92.19% and 91.23% for PEDV, PDCoV and SADS-CoV respectively. In summary, the established microfluidic-RT-LAMP chip detection system could satisfy the demanding in field diagnoses, which was suitable for promotion in remote areas due to its fast, portable and cost-effective characters.


Subject(s)
Coronavirus/genetics , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , Alphacoronavirus/genetics , Alphacoronavirus/isolation & purification , Animals , Coronavirus/isolation & purification , Diarrhea/diagnosis , Diarrhea/veterinary , Diarrhea/virology , Feces/virology , Lab-On-A-Chip Devices , Limit of Detection , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Systems , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/isolation & purification , RNA, Viral/metabolism , Reproducibility of Results , Sensitivity and Specificity , Swine
19.
Cell Syst ; 11(1): 102-108.e3, 2020 07 22.
Article in English | MEDLINE | ID: covidwho-610157

ABSTRACT

SARS-CoV-2 genomic and subgenomic RNA (sgRNA) transcripts hijack the host cell's machinery. Subcellular localization of its viral RNA could, thus, play important roles in viral replication and host antiviral immune response. We perform computational modeling of SARS-CoV-2 viral RNA subcellular residency across eight subcellular neighborhoods. We compare hundreds of SARS-CoV-2 genomes with the human transcriptome and other coronaviruses. We predict the SARS-CoV-2 RNA genome and sgRNAs to be enriched toward the host mitochondrial matrix and nucleolus, and that the 5' and 3' viral untranslated regions contain the strongest, most distinct localization signals. We interpret the mitochondrial residency signal as an indicator of intracellular RNA trafficking with respect to double-membrane vesicles, a critical stage in the coronavirus life cycle. Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.


Subject(s)
Betacoronavirus/genetics , Cell Nucleolus/virology , Coronavirus Infections/virology , Mitochondria/virology , Pneumonia, Viral/virology , RNA, Viral/metabolism , Betacoronavirus/metabolism , Cell Nucleolus/metabolism , Databases, Genetic , Genome, Viral , Humans , Machine Learning , Mitochondria/metabolism , Models, Genetic , Pandemics , RNA, Viral/genetics
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