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1.
Pathogens ; 11(8), 2022.
Article in English | Web of Science | ID: covidwho-2023970

ABSTRACT

Feline infectious peritonitis (FIP) virus is the most common infectious cause of uveitis in cats. Confirmatory diagnosis is usually only reached at postmortem examination. The relationship between the histologic inflammatory pattern, which depends on the stage of the disease, and the likelihood of detection of the viral antigen and/or RNA has not been investigated. We hypothesized that viral detection rate by either immunohistochemistry, in situ hybridization or RT-qPCR is dependent upon the predominant type of uveal inflammatory response (i.e., pyogranulomatous vs. plasmacytic). Thus, the aims of this study were to evaluate cases of FIP-induced uveitis, localize the viral antigen and RNA, and assess the relationship between the inflammatory pattern (macrophage- vs. plasma cell-rich) and the likelihood of detecting the FIP antigen and/or RNA. We evaluated 30 cats with FIP-induced uveitis. The viral antigen and/or RNA were detected within uveal macrophages in 11/30 cases, of which 8 tested positive by RT-qPCR. Correlation analysis determined a weak to moderate but significant negative correlation between the degree of plasmacytic uveal inflammation and the likelihood of detecting the FIP antigen and RNA. This study suggests that predominance of plasmacytic inflammation in cases of FIP uveitis reduces the odds of a confirmatory diagnosis through the viral detection methods available.

2.
25th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2021 ; : 865-866, 2021.
Article in English | Scopus | ID: covidwho-2012793

ABSTRACT

Rolling Circle Amplification (RCA) has shown significant potential for pathogen diagnostics providing high specificity and sensitivity combined with relatively low temperature (<37 °C) isothermal amplification. In the context of the ongoing COVID-19 pandemic, we report the development of an RCA-based method allowing direct detection of SARS-CoV-2 RNA in microfluidics. The viral RNA was hybridized to biotinylated oligos and L-probes in solution, enriched in a microchannel and subsequently amplified in situ using padlock probes against the L-probes. This method allowed the detection of 1x103 viral copies/μL within 90 minutes of amplification, demonstrating an alternative approach to current isothermal amplification methods. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

3.
25th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2021 ; : 849-850, 2021.
Article in English | Scopus | ID: covidwho-2012644

ABSTRACT

Wastewater testing for SARS-CoV-2 has emerged as a promising tool for disease surveillance in aggregate populations. We present a novel method to rapidly extract, concentrate, and amplify viral RNA from wastewater using Exclusion-based Sample Preparation (ESP) and RT-PCR. This technology identified potential outbreaks of SARS-CoV-2 at University of Kentucky dormitories, resulting in targeted clinical testing and quarantine procedures. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

4.
25th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2021 ; : 1441-1442, 2021.
Article in English | Scopus | ID: covidwho-2012360

ABSTRACT

We report the development of an electrochemical sensor platform for ultrasensitive and rapid detection of SARS-CoV-2 viral RNA that integrates loop-mediated isothermal amplification (LAMP), CRISPR-based detection, and anti-fouling nanocomposite coating. By integrating LAMP amplification with CRISPR, we achieved ultrasensitive detection of SARS-CoV-2 RNA at levels as low as 5 copies µL-1. Data from this electrochemical diagnostic platform was comparable to traditional RT-PCR methodology in a fraction of the time, at low cost, and without requiring laboratory space. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

6.
Front Cell Infect Microbiol ; 12: 887800, 2022.
Article in English | MEDLINE | ID: covidwho-1987470

ABSTRACT

The single-stranded viral RNA (ssvRNA) known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19 can be effectively inactivated by a number of natural ribonucleic acid-based host cell defenses. One of the most important of these defenses includes the actions of a class of small non-coding RNAs (sncRNAs) known as microRNAs (miRNAs). Via base-pair complementarity miRNAs are capable of specifically targeting ssvRNA sequences such as SARS-CoV-2 promoting its inactivation and neutralization. RNA-sequencing and bioinformatics analysis indicate that multiple naturally-occurring human miRNAs have extensive complementarity to the SARS-CoV-2 ssvRNA genome. Since miRNA abundance, speciation, and complexity vary significantly amongst human individuals, this may in part explain the variability in the innate-immune and pathophysiological response of different individuals to SARS-CoV-2 and overall susceptibility to ssvRNA-mediated viral infection.


Subject(s)
COVID-19 , MicroRNAs , Humans , Immune System , MicroRNAs/genetics , SARS-CoV-2/genetics
7.
Journal of Water and Health ; : 20, 2022.
Article in English | Web of Science | ID: covidwho-1978926

ABSTRACT

Since infected persons shed SARS-CoV-2 in faeces before symptoms appear, environmental surveillance (ES) may serve as an early warning system (EWS) for COVID-19 and new variants of concern. The ES of SARS-CoV-2 has been widely reviewed;however, its effectiveness as an EWS for SARS-CoV-2 in terms of timeliness, sensitivity and specificity has not been systematically assessed. We conducted a systematic review to identify and synthesise evidence on the ES of SARS-CoV-2 as an EWS to evaluate the added value for public health. Of 1,014 studies identified, we considered 29 for a qualitative synthesis of the timeliness of ES as an EWS for COVID-19, while six studies were assessed for the ability to detect new variants and two for both aims. The synthesis indicates ES may serve as an EWS of 1-2 weeks. ES could complement clinical surveillance for SARS-CoV-2;however, its cost-benefit value for public health decisions needs to be assessed based on the stage of the pandemic and resources available. Studies focusing methodological knowledge gaps as well as how to use and interpret ES signals for public health actions are needed, as is the sharing of knowledge within countries/areas with long experience of such surveillance.

8.
Int J Mol Sci ; 23(15)2022 Jul 27.
Article in English | MEDLINE | ID: covidwho-1969291

ABSTRACT

Influenza virus and coronavirus are two important respiratory viruses, which often cause serious respiratory diseases in humans and animals after infection. In recent years, highly pathogenic avian influenza virus (HPAIV) and SARS-CoV-2 have become major pathogens causing respiratory diseases in humans. Thus, an in-depth understanding of the relationship between viral infection and host innate immunity is particularly important to the stipulation of effective control strategies. As the first line of defense against pathogens infection, innate immunity not only acts as a natural physiological barrier, but also eliminates pathogens through the production of interferon (IFN), the formation of inflammasomes, and the production of pro-inflammatory cytokines. In this process, the recognition of viral pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) is the initiation and the most important part of the innate immune response. In this review, we summarize the roles of RNA sensors in the host innate immune response to influenza virus and coronavirus infections in different species, with a particular focus on innate immune recognition of viral nucleic acids in host cells, which will help to develop an effective strategy for the control of respiratory infectious diseases.


Subject(s)
COVID-19 , Influenza A virus , Animals , Humans , Immunity, Innate , Pathogen-Associated Molecular Pattern Molecules , RNA , SARS-CoV-2
9.
AMB Express ; 12(1): 70, 2022 Jun 09.
Article in English | MEDLINE | ID: covidwho-1968602

ABSTRACT

A worldwide shortage of molecular biology consumables is in surge. This includes filter tips, nucleic acid purification kits, polymerases, reverse-transcriptase, and different types of reagents which are included in viral diagnostic kits. In developing countries, the problem is even worse, since there is few capital enterprise to adopt this kind of industry. So, our aim is to develop a suitable, functional, comparable to commercial ones, and affordable in-house protocol to purify viral RNA. We sought some published and commercial RNA purification solutions to set-up an in-house protocol for viral RNA extraction. Solution was prepared accordingly. Also, LPA (linearized polyacrylamide) carrier was evaluated. The whole setting of in-house solutions with addition of LPA carrier was compared to QIAamp viral RNA minikit solutions. Our results showed that linearized polyacrylamide (LPA) carrier in homemade solutions is comparable to poly A carrier which is used in the most commercial kit. In addition, the whole setting of RNA purification solutions did achieve the purpose of viral RNA purification. Also, the result was confirmed using sputum of a Sars-Cov2 infected patient. Our experiments did end up with an affordable homemade solutions for viral RNA purification.

10.
Sci Total Environ ; 847: 157547, 2022 Jul 22.
Article in English | MEDLINE | ID: covidwho-1956326

ABSTRACT

Wastewater based epidemiology (WBE) has emerged as a strategy to identify, locate, and manage outbreaks of COVID-19, and thereby possibly prevent surges in cases, which overwhelm local to global health care networks. The WBE process is based on assaying municipal wastewater for molecular markers of the SARS-CoV-2 virus. Standard processes for purifying viral RNA from municipal wastewater are often time-consuming and require the handling of large quantities of wastewater, negatively affecting throughput, timely reporting, and safety. We demonstrate here an automated, faster system to purify viral RNA from smaller volumes of wastewater but with increased sensitivity for detection of SARS-CoV-2 markers. We document the effectiveness of this new approach by way of comparison to the PEG/NaCl/Qiagen method prescribed by the State of Michigan for SARS-CoV-2 wastewater monitoring and show its application to several Detroit sewersheds. Specifically, compared to the PEG/NaCl/Qiagen method, viral RNA purification using the PerkinElmer Chemagic™ 360 lowered handling time, decreased the amount of wastewater required by ten-fold, increased the amount of RNA isolated per µl of final elution product by approximately five-fold, and effectively removed ddPCR inhibitors from most sewershed samples. For detection of markers on the borderline of viral detectability, we found that use of the Chemagic™ 360 enabled the measurement of viral markers in a significant number of samples for which the result with the PEG/NaCl/Qiagen method was below the level of detectability. The improvement in detectability of the viral markers might be particularly important for early warning to public health authorities at the beginning of an outbreak. Applied to sewersheds in Detroit, the technique enabled more sensitive detection of SARS-CoV-2 markers with good correlation between wastewater signals and COVID-19 cases in the sewersheds. We also discuss advantages and disadvantages of several automated RNA purification systems, made by Promega, PerkinElmer, and ThermoFisher.

11.
Front Mol Biosci ; 9: 893067, 2022.
Article in English | MEDLINE | ID: covidwho-1952440

ABSTRACT

Identifying human proteins that interact with SARS-CoV-2 genome is important to understand its replication and to identify therapeutic strategies. Recent studies have unveiled protein interactions of SARS-COV-2 in different cell lines and through a number of high-throughput approaches. Here, we carried out a comparative analysis of four experimental and one computational studies to characterize the interactions of SARS-CoV-2 genomic RNA. Although hundreds of interactors have been identified, only twenty-one appear in all the experiments and show a strong propensity to bind. This set of interactors includes stress granule forming proteins, pre-mRNA regulators and elements involved in the replication process. Our calculations indicate that DDX3X and several editases bind the 5' end of SARS-CoV-2, a regulatory region previously reported to attract a large number of proteins. The small overlap among experimental datasets suggests that SARS-CoV-2 genome establishes stable interactions only with few interactors, while many proteins bind less tightly. In analogy to what has been previously reported for Xist non-coding RNA, we propose a mechanism of phase separation through which SARS-CoV-2 progressively sequesters human proteins hijacking the host immune response.

12.
DETRITUS ; 19:94-103, 2022.
Article in English | Web of Science | ID: covidwho-1939694

ABSTRACT

The effect of the COVID-19 pandemic on medical waste EWC/EURAL code 180103* (infectious medical waste) and 180104 (non-infectious medical waste) was investigated in 6 university hospitals and 6 general hospitals. Data on the number of in-hospital patients and on quantity and volume of waste were obtained during 2019 (control period) and in 2020 up to March 2021 (COVID-19 period) for the hospitals, from the waste managing company, and from the regional destruction facility. The presence of SARS-CoV-2 on the surface of waste recipients was analyzed using RTPCR. We found that the effect of the pandemic on the total weight of waste is limited during the first wave (March 2020), while during the second wave, the quantity of waste type 180103* increased. The main effect is a nearly doubling of the volume of waste during both waves caused by the use of cardboard hospital boxes with a yellow inner plastic bag. We demonstrated that the average weight of these cardboard boxes generated for the treatment of COVID-19 patients is significantly lower compared to the weight of the waste from non-COVID-19 patients. COVID-19-related health care activities caused a weight increase of the 180103* waste from historical data (0.2-1.4 kg/day/bed) up to 5-8 kg/day/bed. RT-PCR analysis of swabs demonstrated the absence of viral RNA on personal protection materials and on the surface of recipients containing the waste. We conclude that COVID-19-related hospital waste is predominantly of the EWC 180104 type.

13.
Clinical Laboratory ; : 11, 2022.
Article in English | Web of Science | ID: covidwho-1887317

ABSTRACT

Background: The outbreak of SARS-CoV-2 lead to a worldwide pandemic which poses substantial challenges to public health. Methods: We enrolled 102 consecutive recovered patients with laboratory-confirmed SARS-CoV-2 infection. Epidemiological and demographic characteristics, temporal dynamic profiles of laboratory tests and findings on chest CT radiography, and clinical outcomes were collected and analyzed. Results: Independent risk factors for prolonged fever, viral RNA shedding or radiologic recovery included age of more than 44 years, female gender, having symptoms of cough and fever, a delay from the symptom onset to hospitalization of more than 3 days, a lower CD4 count of less than 500/mu L on admission, and severe or critical illness in hospitalization. The estimated median time from symptom onset was 6.4 (5.5 -7.4) days to peak viral load, 9.1 (7.9 -10.4) days to afebrile, 8 (6.7 -9.4) days to worst radiologic finding, 12.7 (11.2 -14.3) days to viral RNA negativity, and 26.7 (23.8 -29.9) days to radiologic resolution. This study included the entire cross-section of patients seen in our clinical practice and reflected the real-world situation. Conclusions: These findings provide the rationale for strategies of active symptom monitoring, timing of quarantine and antiviral interventions, and duration of radiologic follow-up in patients with COVID-19.

14.
Anal Chim Acta ; 1212: 339909, 2022 Jun 15.
Article in English | MEDLINE | ID: covidwho-1821092

ABSTRACT

Diagnosis of SARS-CoV-2 infection through rapid, accurate, and sensitive testing is the most important and fundamental step in coping with the COVID-19 epidemic. We have developed a sensitive fluorometric assay to detect SARS-CoV-2 viral RNA without thermal cycling. This assay system, based on tandem isothermal gene amplification (TIGA), is composed of ternary rolling circle amplification (t-RCA) and subsequent strand displacement amplification (SDA) coupled with G-quadruplex-generating RCA (SDA/GQ-RCA). Without the need to convert viral RNA into cDNA, viral RNA forms a ternary complex composed of hairpin primer (HP) and dumbbell padlock DNA during the t-RCA process. t-RCA generates a long chain of single-stranded DNA (ssDNA) with tandemly repeated hairpin structures that are subjected to SDA. SDA produces multiple short ssDNAs from t-RCA products, which then serve as primers for the second RCA reaction. A long ssDNA harboring repeated copies of the G-quadruplex is produced in the second round of RCA. Emission of enhanced fluorescence by thioflavin T, which intercalates into the G-quadruplex, allows fluorometric detection of amplified viral genes. This fluorometric analysis sensitively detected SARS-CoV-2 RNA as low as 5.9 aM, with a linear range between 0.2 fM and 200 fM within 1 h. Hence, this isothermal gene amplification method without reverse transcription of viral RNA can be applied to diagnose COVID-19 with high sensitivity and accuracy as an alternative to current PCR-based diagnosis.


Subject(s)
COVID-19 , Reverse Transcription , COVID-19/diagnosis , DNA, Single-Stranded , Gene Amplification , Humans , RNA, Viral/genetics , SARS-CoV-2/genetics
15.
International Journal of Biomathematics ; 15(4):1-27, 2022.
Article in English | Academic Search Complete | ID: covidwho-1816791
16.
J Biol Chem ; 298(5): 101924, 2022 05.
Article in English | MEDLINE | ID: covidwho-1778266

ABSTRACT

The genomes of RNA viruses present an astonishing source of both sequence and structural diversity. From intracellular viral RNA-host interfaces to interactions between the RNA genome and structural proteins in virus particles themselves, almost the entire viral lifecycle is accompanied by a myriad of RNA-protein interactions that are required to fulfill their replicative potential. It is therefore important to characterize such rich and dynamic collections of viral RNA-protein interactions to understand virus evolution and their adaptation to their hosts and environment. Recent advances in next-generation sequencing technologies have allowed the characterization of viral RNA-protein interactions, including both transient and conserved interactions, where molecular and structural approaches have fallen short. In this review, we will provide a methodological overview of the high-throughput techniques used to study viral RNA-protein interactions, their biochemical mechanisms, and how they evolved from classical methods as well as one another. We will discuss how different techniques have fueled virus research to characterize how viral RNA and proteins interact, both locally and on a global scale. Finally, we will present examples on how these techniques influence the studies of clinically important pathogens such as HIV-1 and SARS-CoV-2.


Subject(s)
High-Throughput Nucleotide Sequencing , Proteins , RNA, Viral , HIV-1/genetics , HIV-1/metabolism , Host Microbial Interactions , Humans , Proteins/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics
17.
Drug Des Devel Ther ; 16: 827-841, 2022.
Article in English | MEDLINE | ID: covidwho-1775529

ABSTRACT

The aim of this report is to review the literature and shed light on the uncertainties surrounding the use of antiviral agents in general and remdesivir in COVID-19 patients. This review evaluated a battery of antiviral compounds and their effectiveness in the treatment of COVID-19 since the beginning of the pandemic. Remdesivir is the only antiviral approved by the EMA and FDA for the treatment of SARS-CoV-2 infection. This work extensively reviews remdesivir data generated from clinical trials and observational studies, paying attention to the most recent data, and focusing on outcomes to give readers a more comprehensive understanding of the results. This review also discusses the recommendations issued by official bodies during the pandemic in the light of the current knowledge. The use of remdesivir in the treatment of SARS-CoV-2 infection is justified because a virus is the causative agent that triggers the inflammatory responses and its consequences. More trials are needed to improve the management of this disease.


Subject(s)
COVID-19 , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Humans , SARS-CoV-2 , Virus Replication
18.
J Med Virol ; 94(7): 3133-3137, 2022 07.
Article in English | MEDLINE | ID: covidwho-1739184

ABSTRACT

Clinicians are facing several challenges in tackling coronavirus disease 2019 (COVID-19); one issue is prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Here, we describe a case of SARS-CoV-2 infection in a young immunocompetent patient with a virological course lasting for 71 days. Following antiviral treatment, but no additional glucocorticoid or interferon therapy, the patient recovered from COVID-19 pneumonia (moderate). Detection of viral RNA via throat swabs showed negative results. However, the viral RNA reappeared and persisted in stool samples for an additional 27 days, while the patient remained asymptomatic and exhibited no abnormal signs. This case indicates that SARS-CoV-2 can result in a prolonged fecal RNA shedding, even in an immunocompetent patient with zero exposure to immunosuppressive therapies.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Feces , Humans , RNA, Viral/genetics , Virus Shedding
19.
Aerosol and Air Quality Research ; 22(2), 2022.
Article in English | Scopus | ID: covidwho-1732359

ABSTRACT

Airborne transmission of COVID-19 plays an important role for the pandemic. However, nucleic acid based evidence of direct association of COVID-19 with environmental contamination is lacking. Here, we investigated a COVID-19 outbreak with two fast food employees infected, in which a traveler despite of a 14-day quarantine turned positive after check in with a hotel, using environmental SARS-CoV-2 sampling, epidemiological tracing, viral RNA sequence as well as surveillance method. Out of 25 positive environmental air and surface swab samples (N = 237) collected, SARS-CoV-2 was found to have remained airborne (5640–7840 RNA copies m–3 ) for more than 4 days in a female washroom. After aging for 5 days in the air, no viable virus was detected. The traveler did not have any contacts with the two employees;however, genome sequencing showed that SARS-CoV-2 variants from three patients and two environmental surface samples belonged to 20B viral clade, sharing a nucleic acid identity of more than 99.9%. We concluded that the outbreak was triggered by SARS-CoV-2 contaminated environments, where the employees inhaled the virus from the air or touching facility surfaces where the traveler did not have any physical contacts with. © The Author(s).

20.
Pharmaceuticals (Basel) ; 15(2)2022 Jan 18.
Article in English | MEDLINE | ID: covidwho-1715602

ABSTRACT

Hand-foot-and-mouth disease (HFMD) caused by human enterovirus A71 (EV-A71) infection has been associated with severe neurological complications. With the lack of an internationally approved antiviral, coupled with a surge in outbreaks globally, EV-A71 has emerged as a neurotropic virus of high clinical importance. Andrographolide has many pharmacological effects including antiviral activity and its derivative, andrographolide sulfonate, has been used in China clinically to treat EV-A71 infections. This study sought to identify novel andrographolide derivatives as EV-A71 inhibitors and elucidate their antiviral mode of action. Using an immunofluorescence-based phenotypic screen, we identified novel EV-A71 inhibitors from a 344-compound library of andrographolide derivatives and validated them with viral plaque assays. Among these hits, ZAF-47, a quinolinoxy-andrographolide, was selected for downstream mechanistic studies. It was found that ZAF-47 acts on EV-A71 post-entry stages and inhibits EV-A71 protein expression. Subsequent luciferase studies confirm that ZAF-47 targets EV-A71 genome RNA replication specifically. Unsuccessful attempts in generating resistant mutants led us to believe a host factor is likely to be involved which coincide with the finding that ZAF-47 exhibits broad-spectrum antiviral activity against other enteroviruses (CV-A16, CV-A6, Echo7, CV-B5, CV-A24 and EV-D68). Furthermore, ZAF-46 and ZAF-47, hits from the screen, were derivatives of the same series containing quinolinoxy and olefin modifications, suggesting that an andrographolide scaffold mounted with these unique moieties could be a potential anti-EV-A71/HFMD strategy.

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