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1.
ACS Synth Biol ; 11(5): 1811-1823, 2022 May 20.
Article in English | MEDLINE | ID: covidwho-1815478

ABSTRACT

Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , Mutation , SARS-CoV-2/genetics
2.
Front Public Health ; 9: 749388, 2021.
Article in English | MEDLINE | ID: covidwho-1775929

ABSTRACT

Objective: The main aim of this study was to investigate the prevalence and risk factors of adult self-reported allergic rhinitis and asthma in plain lands and hilly areas of Shenmu City in China, and analyze the differences between regions. Methods: The multi-stage stratified random sampling was applied in a cross-sectional survey of adult residents in Shenmu City, from September to December 2019. The unconditional logistic regression analysis was used to screen the influence factors of allergic rhinitis and asthma. Results: 4,706 adults participated in the survey, and 99% (4,655 in 4,706) completed the questionnaires. The prevalence of allergic rhinitis was 25.4%, and the prevalence of asthma was 9.4%. The prevalence of the allergic rhinitis without asthma, asthma without allergic rhinitis, and the combined allergic rhinitis with asthma were 18.9, 2.9, and 6.5%, respectively. The prevalence of allergic rhinitis and asthma existed regional differences. The prevalence of adult self-reported allergic rhinitis was 41.5% in plain lands areas and 22.1% in hilly areas. The prevalence of adult self-reported asthma was 12.8% in plain lands and 8.8% in hilly areas. The prevalence of allergic rhinitis and asthma existed seasonal differences, with the highest prevalence from July to September. The analysis of risk factors showed that higher education [middle and high school (OR 1.72, 95%CI 1.42-2.07); college and above (OR 2.67, 95%CI 1.99-3.59)], comorbidities of other allergic diseases (OR 3.90, 95%CI 3.23-4.70), family history of allergies (OR 2.89, 95%CI 2.36-3.53), and plain lands areas (OR 2.51, 95%CI 2.06-3.05) were the risk factors for the allergic rhinitis without asthma. Aging [40-49 years old (OR 4.29, 95%CI 1.02-18.13); 50-59 years old (OR 5.89, 95%CI 1.40-24.76); ≥60 years old: (OR 6.14, 95%CI 1.41-26.71)], never-smokers (OR 1.66, 95%CI 0.99-2.80), comorbidities of other allergic disorders (OR 2.17, 95%CI 1.42-3.32), and family history of allergies (OR 2.20, 95%CI 1.40-3.47) were the risk factors for the asthma without allergic rhinitis. Advanced age [30-39 years (OR 2.16, 95%CI 1.23-3.82); 40-49 years (OR 2.86, 95%CI 1.56 to 5.25); 50-59 years (OR 2.95, 95%CI 1.58-5.51); ≥60 years old (OR 2.27, 95%CI 1.09-4.72)], higher education [middle and high school (OR 2.23, 95%CI 1.62-3.07); college and above (OR 4.28, 95%CI 2.72-6.74)], non-agricultural workers (OR 1.70, 95%CI 1.18-2.43),never-smokers (OR 2.26, 95%CI 1.51-3.39), comorbidities of other allergic diseases (OR 4.45, 95%CI 3.37-5.88), family history of allergies (OR 5.27, 95%CI 3.98-6.97), and plain lands areas (OR 2.07, 95%CI 1.51-2.86) were the risk factors for the combined allergic rhinitis with asthma. Conclusions: The prevalence of allergic rhinitis and asthma in Shenmu City was relatively high, with regional differences. Genetic and environmental factors were the important risk factors associated with allergic rhinitis and asthma. Our research would provide data support for preventing and controlling allergic rhinitis and asthma in this region in the future, and appropriate prevention and control programs should be formulated according to the characteristics of different regions.


Subject(s)
Asthma , Rhinitis, Allergic , Adult , Asthma/complications , Asthma/epidemiology , China/epidemiology , Cross-Sectional Studies , Humans , Middle Aged , Prevalence , Rhinitis, Allergic/complications , Rhinitis, Allergic/epidemiology , Risk Factors , Self Report
3.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-323582

ABSTRACT

The past twelve years were punctuated by increasingly complex dynamics of the cross-market interdependence and two ``once-in-100-year'' global financial crises, including the 2020 financial contagion through increased physical contagion during the COVID-19 pandemic. This paper develops a non-linear financial contagion network via a dynamic mixture copula-EVT (extreme value theory) model to quantitatively detect and measure the complex nature of financial contagion. Considering the crisis transmission from both macroeconomic fundamentals and investor constraint perspectives, we identify and investigate the dynamics of transmission channels for both 2008 and 2020 financial crises. More specifically, we explore the wealth effect and portfolio rebalancing through the asymmetric tail contagion channels across 22 major emerging and developed stock markets and compare the differences between the two major crises. Moreover, motivated by the V-shape rebound in these two crises, we examine and find evidence that the transmission channels between the market melt-down and melt-up periods are different. Our findings shed new light on the dynamics of crisis transmission mechanisms across countries and provide important implications for international investors and policymakers.

4.
Appl Math Comput ; 421: 126911, 2022 May 15.
Article in English | MEDLINE | ID: covidwho-1620456

ABSTRACT

Dimension governs dynamical processes on networks. The social and technological networks which we encounter in everyday life span a wide range of dimensions, but studies of spreading on finite-dimensional networks are usually restricted to one or two dimensions. To facilitate investigation of the impact of dimension on spreading processes, we define a flexible higher-dimensional small world network model and characterize the dependence of its structural properties on dimension. Subsequently, we derive mean field, pair approximation, intertwined continuous Markov chain and probabilistic discrete Markov chain models of a COVID-19-inspired susceptible-exposed-infected-removed (SEIR) epidemic process with quarantine and isolation strategies, and for each model identify the basic reproduction number R 0 , which determines whether an introduced infinitesimal level of infection in an initially susceptible population will shrink or grow. We apply these four continuous state models, together with discrete state Monte Carlo simulations, to analyse how spreading varies with model parameters. Both network properties and the outcome of Monte Carlo simulations vary substantially with dimension or rewiring rate, but predictions of continuous state models change only slightly. A different trend appears for epidemic model parameters: as these vary, the outcomes of Monte Carlo change less than those of continuous state methods. Furthermore, under a wide range of conditions, the four continuous state approximations present similar deviations from the outcome of Monte Carlo simulations. This bias is usually least when using the pair approximation model, varies only slightly with network size, and decreases with dimension or rewiring rate. Finally, we characterize the discrepancies between Monte Carlo and continuous state models by simultaneously considering network efficiency and network size.

5.
Front Microbiol ; 12: 789062, 2021.
Article in English | MEDLINE | ID: covidwho-1581272

ABSTRACT

Mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have made this virus more infectious. Previous studies have confirmed that non-structural protein 13 (NSP13) plays an important role in immune evasion by physically interacting with TANK binding kinase 1 (TBK1) to inhibit IFNß production. Mutations have been reported in NSP13; hence, in the current study, biophysical and structural modeling methodologies were adapted to dissect the influence of major mutations in NSP13, i.e., P77L, Q88H, D260Y, E341D, and M429I, on its binding to the TBK1 and to escape the human immune system. The results revealed that these mutations significantly affected the binding of NSP13 and TBK1 by altering the hydrogen bonding network and dynamic structural features. The stability, flexibility, and compactness of these mutants displayed different dynamic features, which are the basis for immune evasion. Moreover, the binding was further validated using the MM/GBSA approach, revealing that these mutations have higher binding energies than the wild-type (WT) NSP13 protein. These findings thus justify the basis of stronger interactions and evasion for these NSP13 mutants. In conclusion, the current findings explored the key features of the NSP13 WT and its mutant complexes, which can be used to design structure-based inhibitors against the SARS-CoV-2 new variants to rescue the host immune system.

6.
Microbiol Spectr ; 9(3): e0101721, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1522923

ABSTRACT

A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.


Subject(s)
COVID-19 Testing/methods , COVID-19/virology , CRISPR-Cas Systems , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Alleles , COVID-19/diagnosis , Databases, Nucleic Acid , Humans , Mass Screening , Mutation , Polymerase Chain Reaction , Public Health , Spike Glycoprotein, Coronavirus/genetics
7.
Front Microbiol ; 12: 703145, 2021.
Article in English | MEDLINE | ID: covidwho-1337656

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been continuously mutating since its first emergence in early 2020. These alterations have led this virus to gain significant difference in infectivity, pathogenicity, and host immune evasion. We previously found that the open-reading frame 8 (ORF8) of SARS-CoV-2 can inhibit interferon production by decreasing the nuclear translocation of interferon regulatory factor 3 (IRF3). Since several mutations in ORF8 have been observed, therefore, in the present study, we adapted structural and biophysical analysis approaches to explore the impact of various mutations of ORF8, such as S24L, L84S, V62L, and W45L, the recently circulating mutant in Pakistan, on its ability to bind IRF3 and to evade the host immune system. We found that mutations in ORF8 could affect the binding efficiency with IRF3 based on molecular docking analysis, which was further supported by molecular dynamics simulations. Among all the reported mutations, W45L was found to bind most stringently to IRF3. Our analysis revealed that mutations in ORF8 may help the virus evade the immune system by changing its binding affinity with IRF3.

8.
J Virol Methods ; 292: 114141, 2021 06.
Article in English | MEDLINE | ID: covidwho-1142107

ABSTRACT

In this study, we developed and evaluated a luciferase immunosorbent assay (LISA) for quantitative detection of IgG antibody against SARS-CoV-2 nucleoprotein (NP). Anti-SARS-CoV-2 NP antibody in serum or plasma samples was captured by protein G-coated microtiter plate and detected using the crude cell lysates expressing Nanoluc luciferase (Nluc) enzyme fused with SARS-CoV-2 NP. After the addition of furimazine substrate, the levels of anti-SARS-CoV-2 NP IgG antibody were quantitatively measured as luciferase light units. As expected, SARS-CoV-2 NP showed cross-reactivity with the monoclonal antibodies against SARS-CoV NP, but not MERS-CoV NP-specific monoclonal antibodies or the monoclonal antibodies against SARS-CoV Spike protein. LISA for detecting murine monoclonal antibody against SARS-CoV NP showed a low limit of detection of 0.4 pg/µl and linear detection range from 0.4 pg/µl to 75 pg/µl. Furthermore, LISA had a sensitivity of 71 % when testing COVID-19 patients at the second week post onset and a specificity of 100 % when testing healthy blood donors.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/blood , Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Antibodies, Monoclonal/immunology , Cross Reactions , Humans , Luciferases
9.
Virus Res ; 296: 198350, 2021 04 15.
Article in English | MEDLINE | ID: covidwho-1091608

ABSTRACT

The open reading frame 8 (orf8) is an accessory protein of SARS-CoV-2. It has 121 amino acids with two genotypes, orf8L and orf8S. In this study, we overexpressed the orf8L and orf8S of SARS-CoV-2 as well as the orf8b of SARS-CoV to investigate their roles in the regulation of endoplasmic reticulum (ER) stress and the inhibition of interferon beta (IFNß) production. We found that the two genotypes of SARS-CoV-2 orf8 are capable of inducing ER stress without significant difference by triggering the activating transcription factor 6 (ATF6) and inositol-requiring enzymes 1 (IRE1) branches of the ER stress pathway. However, the third branch of ER stress pathway, i.e. the protein kinase-like ER kinase (PERK), was unaffected by the overexpression of SARS-CoV-2 orf8L or orf8S. Moreover, both orf8L and orf8S of SARS-CoV-2 are capable of down regulating the production of IFNß and interferon-stimulated genes (ISG), ISG15 and ISG56 induced by polyinosinic-polycytidylic acid (poly (I:C)). Moreover, we also found decreased nuclear translocation of Interferon regulatory factor 3 (IRF3), after overexpressing orf8L and orf8S induced by poly (I:C). Our data demonstrated that SARS-CoV-2 orf8 protein could induce ER stress by activating the ATF6 and IRE1 pathways, but not the PERK pathway, and functions as an interferon antagonist to inhibit the production of IFNß. However, these functions appeared not to be affected by the genotypes of SARS-CoV-2 orf8L and orf8S.


Subject(s)
Endoplasmic Reticulum Stress/physiology , Immune Evasion , Interferon-beta/antagonists & inhibitors , Viral Proteins/physiology , Activating Transcription Factor 6/physiology , Endoribonucleases/physiology , HEK293 Cells , Humans , Interferon-beta/biosynthesis , Sequence Alignment , Signal Transduction/physiology , Unfolded Protein Response , Viral Proteins/chemistry , X-Box Binding Protein 1/physiology , eIF-2 Kinase/physiology
11.
SciFinder; 2020.
Preprint | SciFinder | ID: ppcovidwho-4830

ABSTRACT

A review. OBJECTIVE To promote the safety and rationality of traditional Chinese medicine for prevention and treatment of COVID-19 in the elderly. METHODS According to the traditional Chinese medicine (TCM) prevention program of COVID-19 formulated by the various of China, combined with the physiol. characteristics, pathol. conditions, basic diseases and medication principles of the elderly were analyzed. RESULTS This article proposes a rational application strategy of Chinese medicine for the prevention and treatment of COVID-19 in the elderly. CONCLUSION As the key population for the prevention of SARS-CoV-2 infection, the intervention of rational drug use should be strengthened to ensure the safety use of traditional Chinese medicine and reduce adverse reactions in the elderly.

12.
Virology ; 551: 26-35, 2020 12.
Article in English | MEDLINE | ID: covidwho-799506

ABSTRACT

BACKGROUND: SARS-CoV-2 is a novel coronavirus and the cause of COVID-19. More than 80% of COVID-19 patients exhibit mild or moderate symptoms. In this study, we investigated the dynamics of viral load and antibodies against SARS-CoV-2 in a longitudinal cohort of COVID-19 patients with severe and mild/moderate diseases. METHODS: Demographic and clinical information were obtained. Serial samples of blood, nasal and pharyngeal and anal swabs were collected at different time points post-onset. SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies were measured by qRT-PCR and immunoassays, respectively. RESULTS: Respiratory SARS-CoV-2 RNA was detectable in 58.0% (58/100) COVID-19 patients upon admission and lasted for a median of 13 days post-onset. In addition, 5.9% (1/17) and 20.2% (19/94) of the blood and anal swab specimens were positive for SARS-CoV-2 RNA, respectively. Anal viral RNA was more frequently detected in the patients who were positive for viral RNA in the respiratory samples upon admission. Specific anti-SARS-CoV-2 antibody developed within two weeks after onset, reached peak approximately 17 days post-onset and then maintained at relatively high level up to 50 days we analyzed in most patients. However, the levels of antibodies were variable among the patients. High titers of antibodies appeared to be associated with the severity of the disease. Furthermore, viral proteins from different sources showed significant difference of serological sensitivity especially during the first week post-onset. CONCLUSIONS: Our results indicate rapid clearance or self-elimination of viral RNA in about half of the COVID-19 patients upon admission. Viral RNA shedding of SARS-CoV-2 occurred in multiple tissues including the respiratory system, blood, and intestine. Variable levels of specific anti-SARS-CoV-2 antibody may be associated with disease severity. These findings have shed light on viral kinetics and antibody response in COVID-19 patients and provide scientific evidence for infection control and patient management.


Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Adult , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/blood , COVID-19/diagnosis , Female , Humans , Kinetics , Longitudinal Studies , Male , Middle Aged , Nasopharynx/virology , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Viral Load , Virus Shedding
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