A Universal Fluorescent Immunochromatography Assay Based on Quantum Dot Nanoparticles for the Rapid Detection of Specific Antibodies against SARS-CoV-2 Nucleocapsid Protein
Zehui LiAiping WangJingming ZhouYumei ChenHongliang LiuYankai LiuYing ZhangPeiyang DingXifang ZhuChao LiangYanhua QiEnping LiuGaiping Zhang
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Abstract:
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogenic agent leading to COVID-19. Due to high speed of transmission and mutation rates, universal diagnosis and appropriate prevention are still urgently needed. The nucleocapsid protein of SARS-CoV-2 is considered more conserved than spike proteins and is abundant during the virus' life cycle, making it suitable for diagnostic applications. Here, we designed and developed a fluorescent immunochromatography assay (FICA) for the rapid detection of SARS-CoV-2-specific antibodies using ZnCdSe/ZnS QDs-conjugated nucleocapsid (N) proteins as probes. The nucleocapsid protein was expressed in E.coli and purified via Ni-NTA affinity chromatography with considerable concentration (0.762 mg/mL) and a purity of more than 90%, which could bind to specific antibodies and the complex could be captured by Staphylococcal protein A (SPA) with fluorescence displayed. After the optimization of coupling and detecting conditions, the limit of detection was determined to be 1:1.024 × 105 with an IgG concentration of 48.84 ng/mL with good specificity shown to antibodies against other zoonotic coronaviruses and respiratory infection-related viruses (n = 5). The universal fluorescent immunochromatography assay simplified operation processes in one step, which could be used for the point of care detection of SARS-CoV-2-specific antibodies. Moreover, it was also considered as an efficient tool for the serological screening of potential susceptible animals and for monitoring the expansion of virus host ranges.Keywords:
Coronavirus
Ten years after the outbreak of the Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV, which caused the first pandemic of the 21st century, a novel betacoronavirus, Middle East Respiratory Syndrome Coronavirus, MERS-CoV, emerged in the Arabian Peninsula. Its ongoing spread poses a significant threat to public health. The spike (S) protein of coronaviruses mediates viral entry into target cells and is a key determinant of viral tropism and pathogenesis. Understanding the parameters governing MERS-CoV spike (MERS-S)-driven entry might thus yield valuable information on MERS-CoV biology and was the first goal of the present study. In order to facilitate host cell entry, coronavirus S proteins depend on activation by host cell proteases, which are potential targets for therapeutic intervention. Therefore, the second goal of the present thesis was to identify the proteases responsible for MERS-S activation. The coronavirus S protein is the major target for the neutralizing antibody response and experimental systems for MERS-S can be used as diagnostic tools. The final goal of this thesis was thus to investigate the MERS-CoV seroprevalence in Saudi Arabian individuals. A lentiviral vector system was established that allows the analysis of MERS-S-driven host cell entry. With the help of this system, MERS-S was found to mediate entry into a broad spectrum of human cell lines, including cells from lung, kidney and colon which is in concordance with the clinical picture of MERS. Host cell entry was independent of previously described coronavirus entry receptors but was promoted by the endosomal cysteine protease cathepsin L and the transmembrane serine protease TMPRSS2. In contrast, the activity of proprotein convertases was dispensable for MERS-S protein-driven entry. Finally, neutralization of S protein-mediated entry revealed that neutralizing antibodies were absent in sera from patients of the Eastern Province of Saudi Arabia taken between 2010-2011 and 2012, indicating that MERS-CoV infections were rare events before the MERS outbreak in 2012. Collectively, these results provide important insights into the processes governing MERS-CoV entry and shed light onto MERS epidemiology. Furthermore, the demonstration that the protease inhibitor camostat, which is approved for use in humans in Japan, blocks MERS-CoV entry by inhibiting TMPRSS2, might help to establish treatment options for MERS patients.
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2019 novel coronavirus (2019-nCoV) is a new member of coronavirus family that can cause serious respiratory diseases after the emergence of severe acute respiratory syndrome-coronavirus (SARS-CoV) and middle east respiratory syndrome-coronavirus (MERS-CoV). At present, there is no specific antiviral drug targeting 2019-nCoV. In facing of the increasingly serious epidemic of 2019 novel coronavirus pneumonia and the urgent needs in drug treatment strategies, this paper reviewed the current research situation and progress in antiviral treatment for the newly identified disease.
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2019 novel coronavirus; Treatment; Drugs
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2019 novel coronavirus (2019-nCoV) is a new member of coronavirus family that can cause serious respiratory diseases after the emergence of severe acute respiratory syndrome-coronavirus (SARS-CoV) and middle east respiratory syndrome-coronavirus (MERS-CoV). At present, there is no specific antiviral drug targeting 2019-nCoV. In facing of the increasingly serious epidemic of 2019-nCoV pneumonia and the urgent needs in drug treatment strategies, this paper reviewed the current research situation and progress in antiviral treatment for the newly identified disease.
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2019-20 coronavirus outbreak
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Since December 2019, a series of unexplained pneumonia cases have been reported in Hubei Province, China. Scientists have isolated a new type of coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), which has been infected by the virus. The epidemic in China and many countries around the world has attracted global attention. This is the third type of coronavirus discovered after the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). At present, many scholars have conducted a lot of research on it. This article focuses on SARS-CoV. The etiology, epidemiological characteristics, clinical manifestations, imaging examinations, laboratory examinations, diagnosis and treatment of CoV-2 are reviewed.
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Etiology
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Currently, the expansion of the novel human respiratory coronavirus (known as SARS-CoV-2 [severe acute respiratory syndrome coronavirus 2], COVID-2019 [coronavirus disease 2019], or 2019-nCoV [2019 novel coronavirus]) has stressed the need for therapeutic alternatives to alleviate and stop this new epidemic. The previous epidemics of infections by high-morbidity human coronaviruses, such as SARS-CoV in 2003 and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, prompted the characterization of compounds that could be potentially active against the currently emerging novel coronavirus, SARS-CoV-2.
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Coronavirus is RNA virus, which exists widely among human and animals. Human coronavirus (HCoVs) is a common pathogen of community respiratory tract infection. Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and new coronavirus 2019-nCoV had caused 3 human outbreaks during 20 years. Children are generally susceptible to coronavirus. Compared with adults, children with viral infection tend to have age-related characteristics due to their relatively physiological immaturity. The characteristics of community acquired infection of HCoVs in children, Severe acute respiratory syndrome (SARS) in children, Middle East respiratory syndrome (MERS) in children were summarized in this paper. The diagnosis and treatment of 2019 novel coronavirus disease (COVID-19) in children were concluded, based on the official COVID-19 programs, expert suggestion of COVID-19 and limited data of 2019-nCoV childhood infection csaes published in China within 2 month recently.
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Coronavirus; Children; 2019-nCoV; Novel coronavirus pneumonia
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In 2012, a novel coronavirus, initially named as human coronavirus EMC (HCoV-EMC) but recently renamed as Middle East respiratory syndrome human coronavirus (MERS-CoV), was identified in patients who suffered severe acute respiratory infection and subsequent renal failure that resulted in death. Ongoing epidemiological investigations together with retrospective studies have found 61 laboratory-confirmed cases of infection with this novel coronavirus, including 34 deaths to date. This novel coronavirus is culturable and two complete genome sequences are now available. Furthermore, molecular detection and indirect immunofluorescence assay have been developed. The present paper summarises the limited recent advances of this novel human coronavirus, including its discovery, genomic characterisation and detection.
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In the past 17 years, three novel coronaviruses have caused severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the coronavirus disease 2019 (COVID-19). As emerging infectious diseases, they were characterized by their novel pathogens and transmissibility without available clinical drugs or vaccines. This is especially true for the newly identified COVID-19 caused by SARS coronavirus 2 (SARS-CoV-2) for which, to date, no specific antiviral drugs or vaccines have been approved. Similar to SARS and MERS, the lag time in the development of therapeutics is likely to take months to years. These facts call for the development of broad-spectrum anti-coronavirus drugs targeting a conserved target site. This review will systematically describe potential broad-spectrum coronavirus fusion inhibitors, including antibodies, protease inhibitors, and peptide fusion inhibitors, along with a discussion of their advantages and disadvantages.
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Ten years after the outbreak of the Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV, which caused the first pandemic of the 21st century, a novel betacoronavirus, Middle East Respiratory Syndrome Coronavirus, MERS-CoV, emerged in the Arabian Peninsula. Its ongoing spread poses a significant threat to public health. The spike (S) protein of coronaviruses mediates viral entry into target cells and is a key determinant of viral tropism and pathogenesis. Understanding the parameters governing MERS-CoV spike (MERS-S)-driven entry might thus yield valuable information on MERS-CoV biology and was the first goal of the present study. In order to facilitate host cell entry, coronavirus S proteins depend on activation by host cell proteases, which are potential targets for therapeutic intervention. Therefore, the second goal of the present thesis was to identify the proteases responsible for MERS-S activation. The coronavirus S protein is the major target for the neutralizing antibody response and experimental systems for MERS-S can be used as diagnostic tools. The final goal of this thesis was thus to investigate the MERS-CoV seroprevalence in Saudi Arabian individuals.
A lentiviral vector system was established that allows the analysis of MERS-S-driven host cell entry. With the help of this system, MERS-S was found to mediate entry into a broad spectrum of human cell lines, including cells from lung, kidney and colon which is in concordance with the clinical picture of MERS. Host cell entry was independent of previously described coronavirus entry receptors but was promoted by the endosomal cysteine protease cathepsin L and the transmembrane serine protease TMPRSS2. In contrast, the activity of proprotein convertases was dispensable for MERS-S protein-driven entry. Finally, neutralization of S protein-mediated entry revealed that neutralizing antibodies were absent in sera from patients of the Eastern Province of Saudi Arabia taken between 2010-2011 and 2012, indicating that MERS-CoV infections were rare events before the MERS outbreak in 2012. Collectively, these results provide important insights into the processes governing MERS-CoV entry and shed light onto MERS epidemiology. Furthermore, the demonstration that the protease inhibitor camostat, which is approved for use in humans in Japan, blocks MERS-CoV entry by inhibiting TMPRSS2, might help to establish treatment options for MERS patients.
Coronavirus
Betacoronavirus
Tissue tropism
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