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Publication
Journal: Advances in Virology
August/23/2012
Abstract
The human coronaviruses (HCoVs) HCoV-NL63 and HCoV-HKU1 are two recently discovered coronaviruses that circulate widely and are associated with acute respiratory infections (ARI). We detected HCoV-NL63 and HCoV-HKU1 in specimens collected from May 2008 to March 2010 from patients with ARI aged <7.75 years of age attending the Beijing Children's Hospital. Thirty-two (8.4%) and 57 (14.9%) of 382 specimens tested positive for HCoV-NL63 and HCoV-HKU1, respectively, by real-time RT-PCR. Use of a Luminex xTAG RVP Fast kit showed that coinfection with respiratory syncytial virus and parainfluenza 3 virus was common among patients infected with either virus type. In HCoV-HKU1-infected patients, the predominant clinical symptoms were cough, fever, and expectoration. In HCoV-NL63-infected patients they were cough, fever, and rhinorrhea. Phylogenetic studies showed that the HCoV-HKU1 nucleoprotein gene was relatively conserved compared to NCBI reference sequences, while the 1ab gene of HCoV-NL63 showed more variation.
Publication
Journal: Journal of Infectious Diseases
March/21/2006
Abstract
We investigated whether infection with a novel human coronavirus (HCoV), called "New Haven coronavirus" (HCoV-NH)--which is similar to and likely represents the same species as another novel HCoV, HCoV-NL63--is associated with Kawasaki disease (KD) in Taiwan. Fifty-three patients with KD were enrolled in our study. Serum, peripheral-blood mononuclear cells, nasopharyngeal aspirates, throat swabs, and rectal swabs from these patients were assayed for HCoV-NL63 by real-time reverse-transcriptase (RT) polymerase chain reaction (PCR), and the throat swabs, nasopharyngeal aspirates, and rectal swabs were also assayed for HCoV-NH by RT-PCR. All PCR results were negative for both HCoV-NL63 and HCoV-NH; therefore, we did not find any association between HCoV-NH infection and KD in Taiwan.
Publication
Journal: The open virology journal
July/14/2011
Abstract
Even though coronavirus infection of humans is not normally associated with severe diseases, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome showed that highly pathogenic coronaviruses can enter the human population. Shortly thereafter, in Holland in 2004, another novel human coronavirus (HCoV-NL63) was isolated from a seven-month old infant suffering from respiratory symptoms. This virus has subsequently been identified in various countries, indicating a worldwide distribution. HCoV-NL63 has been shown to infect mainly children and the immunocommpromised, who presented with either mild upper respiratory symptoms (cough, fever and rhinorrhoea) or more serious lower respiratory tract involvement such as bronchiolitis and croup, which was observed mainly in younger children. In fact, HCoV-NL63 is the aetiological agent for up to 10% of all respiratory diseases. This review summarizes recent findings of human coronavirus HCoV-NL63 infections, including isolation and identification, phylogeny and taxonomy, genome structure and transcriptional regulation, transmission and pathogenesis, and detection and diagnosis.
Publication
Journal: Viruses
September/10/2012
Abstract
HCoV-NL63 is a recently identified respiratory virus. Its pathogenesis has not been fully unraveled because an animal model is currently lacking. Here we examined whether rhesus macaques encounter HCoV-NL63 infections during life, by examining the levels of antibodies to HCoV-NL63 in time. The animals were followed for 7 up till 19 years, and in three animals we observed a steep rise in antibodies during follow up, indicative of a natural infection with HCoV-NL63.
Publication
Journal: Virus Research
July/16/2012
Abstract
Recent research has shown that Coronavirus (CoV) replication depends on active immunophilin pathways. Here we demonstrate that the drug FK506 (Tacrolimus) inhibited strongly the growth of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E at low, non-cytotoxic concentrations in cell culture. As shown by plaque titration, qPCR, Luciferase- and green fluorescent protein (GFP) reporter gene expression, replication was diminished by several orders of magnitude. Knockdown of the cellular FK506-binding proteins FKBP1A and FKBP1B in CaCo2 cells prevented replication of HCoV-NL63, suggesting the requirement of these members of the immunophilin family for virus growth.
Publication
Journal: Journal of Virology
November/14/2018
Abstract
Human coronaviruses (HCoV) are recognized respiratory pathogens for which accumulating evidence indicates that in vulnerable patients, the infection can cause more severe pathologies. HCoVs are not always confined to the upper respiratory tract and can invade the CNS upon still unclear circumstances. HCoV-induced neuropathologies in human are difficult to diagnose early enough to allow therapeutic interventions. Making use of our already described animal model of HCoV neuropathogenesis, we describe the route of neuropropagation from the nasal cavity to the olfactory bulb, piriform cortex then brainstem. We identified neuron-to-neuron propagation as one underlying mode of virus spreading in cell culture. Our data demonstrate that both passive diffusion of released viral particles and axonal transport are valid propagation strategies used by the virus. We describe for the first time the presence along axons of viral platforms whose static dynamism are reminiscent of viral assembly sites. We further revealed that HCoV-OC43 modes of propagation could be modulated by selected HCoV-OC43 proteins and axonal transport. Our work, therefore, identifies processes that may govern the severity and nature of HCoV-OC43 neuropathogenesis and will make possible the development of therapeutic strategies to prevent occurrences.IMPORTANCE Coronaviruses may invade the CNS, disseminate and participate in the induction of neurological diseases. Their neuropathogenicity is being increasingly recognized in humans, and the presence and persistence of human coronaviruses (HCoV) in human brains was proposed to cause long-term sequelae. Using our mouse model relying on natural susceptibility to HCoV-OC43 and neuronal cell cultures, we have defined the most relevant path taken by HCoV-OC43 to access and spread to and within the CNS toward the brainstem and spinal cord and studied in cell culture the underlying modes of intercellular propagation to better understand its neuropathogenesis. Our data suggest that the axonal transport governs HCoV-OC43 egress in the CNS leading to exacerbate neuropathogenesis. Exploiting knowledge on neuroinvasion and dissemination will enhance our ability to control viral infection within the CNS as it will shed light on underlying mechanisms of neuropathogenesis and uncover potential "druggable" molecular virus-host interfaces.
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Publication
Journal: Viruses
November/14/2018
Abstract
Human coronaviruses (HCoVs) cause mild to severe respiratory diseases. Six types of HCoVs have been discovered, the most recent one termed the Middle East respiratory syndrome coronavirus (MERS-CoV). The aim of this study is to monitor the circulation of HCoV types in the population during 2015⁻2016 in Israel. HCoVs were detected by real-time PCR analysis in 1910 respiratory samples, collected from influenza-like illness (ILI) patients during the winter sentinel influenza survey across Israel. Moreover, 195 HCoV-positive samples from hospitalized patients were detected during one year at Soroka University Medical Center. While no MERS-CoV infections were detected, 10.36% of patients in the survey were infected with HCoV-OC43 (43.43%), HCoV-NL63 (44.95%), and HCoV-229E (11.62%) viruses. The HCoVs were shown to co-circulate with respiratory syncytial virus (RSV) and to appear prior to influenza virus infections. HCoV clinical symptoms were more severe than those of RSV infections but milder than influenza symptoms. Hospitalized patients had similar HCoV types percentages. However, while it was absent from the public winter survey, 22.6% of the patients were HCoV-HKU1 positives, mainly during the spring-summer period.
Related with
Publication
Journal: Methods in Molecular Biology
March/19/2009
Abstract
Calculation of infectious viral titers represents a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for strains 229E and OC43 of human coronavirus (HCoV). An alternative indirect immunoperoxidase assay (IPA) is herein described for the detection and titration of these viruses. Susceptible cells are inoculated with serial logarithmic dilutions of samples in a 96-well plate. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as "Tissue Culture Infectious Dose" (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain replicating virus. This technique is a reliable method for the titration of HCoV in biological samples (cells, tissues, or fluids).
Publication
Journal: Journal of Medical Virology
September/19/2005
Abstract
A new human coronavirus, HCoV-NL63, was associated recently with bronchiolitis. The current study aimed to examine retrospectively stored specimens for the presence of HCoV-NL63 using nested RT-PCR assays targeting the 1a and 1b genes. The study population was composed of patients with acute respiratory disease warranting presentation to Queensland hospitals. HCoV-NL63 was detected in the nasopharyngeal aspirates (NPA) of 16 of 840 specimens representing 766 patients (2%). HCoV-NL63 positive individuals were diagnosed most commonly with lower respiratory tract (LRT) disease (81%). The clinical diagnosis was commonly supported by an abnormal chest X-ray (56%) together with respiratory distress (50%), wheeze (44%), and rales (25%) on first presentation with HCoV-NL63 infection. All patients positive for HCoV-NL63 required admission to hospital. Among 38% of HCoV-NL63 positive specimens a second pathogen was detected. Sequencing of amplicon from gene 1b revealed more than 99% nucleotide homology with the viral type strains while sequencing amplicon from gene 1a permitted the grouping of viral strains. It was shown that HCoV-NL63 is associated with severe LRT disease in an Australian hospital setting during the cooler months of the year. We propose that HCoV-NL63 is a global and seasonal pathogen of both children and adults associated with severe LRT illness.
Publication
Journal: ACS Applied Materials & Interfaces
October/22/2019
Abstract
Therapeutic options for the highly pathogenic human coronavirus (HCoV) infections are urgently needed. Anti-coronavirus therapy is however challenging, as coronaviruses are biologically diverse and rapidly mutating. In this work, the antiviral activity of seven different carbon quantum dots (CQDs) for the treatment of human coronavirus HCoV-229E infections was investigated. The first generation of antiviral CQDs was derived by hydrothermal carbonization from ethylenediamine/citric acid as carbon precursors and post-modified with boronic acid ligands. These nanostructures showed a concentration dependent virus inactivation with an estimated EC50 of 52±8 µg mL-1. CQDs derived from 4-aminophenylboronic acid without any further modification resulted in the second-generation of anti-HCoV nanomaterials with an EC50 lowered to 5.2±0.7 µg mL-1. The underlying mechanism of action of these CQDs revealed to be inhibition of HCoV-229E entry that could be due to interaction of the functional groups of the CQDs with HCoV-229E entry receptors; surprisingly, an equally large inhibition activity was observed at the viral replication step.
Publication
Journal: Journal of Virology
September/29/2008
Abstract
The newly emergent human coronavirus HKU1 (HCoV-HKU1) was first identified in Hong Kong in 2005. Infection by HCoV-HKU1 occurs worldwide and causes syndromes such as the common cold, bronchitis, and pneumonia. The CoV main protease (M(pro)), which is a key enzyme in viral replication via the proteolytic processing of the replicase polyproteins, has been recognized as an attractive target for rational drug design. In this study, we report the structure of HCoV-HKU1 M(pro) in complex with a Michael acceptor, inhibitor N3. The structure of HCoV-HKU1 provides a high-quality model for group 2A CoVs, which are distinct from group 2B CoVs such as severe acute respiratory syndrome CoV. The structure, together with activity assays, supports the relative conservation at the P1 position that was discovered by sequencing the HCoV-HKU1 genome. Combined with structural data from other CoV M(pro)s, the HCoV-HKU1 M(pro) structure reported here provides insights into both substrate preference and the design of antivirals targeting CoVs.
Publication
Journal: Biochemical and Biophysical Research Communications
August/1/2006
Abstract
Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six alpha-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection.
Publication
Journal: Antiviral Research
July/8/2013
Abstract
The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity.
Publication
Journal: Biochemical and Biophysical Research Communications
May/9/2018
Abstract
HCoV-229E spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This protein is composed of an N-terminal receptor-binding domain (S1) and a C-terminal trans-membrane fusion domain (S2). S2 contains a highly conserved heptad repeat 1 and 2 (HR1 and HR2). In this study, the HRs sequences were designed and connected with a flexible linker. The recombinant fusion core protein was crystallized and its structure was solved at a resolution of 2.45 Å. Then we characterized the binding of HR1s and HR2s via both sequence alignment and structural analysis. The overall structures, especially the residues in some positions of HR2 are highly conserved. Fourteen hydrophobic and three polar residues from each HR1 peptide are packed in layers at the coiled-coil interface. These core amino acids can be grouped into seven heptad repeats. Analysis of hydrophobic and hydrophilic interactions between HR2 helix and HR1 helices, shows that the HR1 and HR2 polypeptides are highly complementary in both shape and chemical properties. Furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting membrane fusion, a crucial step of HCoV-229E infection.
Publication
Journal: International Journal of Molecular Sciences
August/8/2018
Abstract
Human coronavirus 229E (HCoV-229E) infection in infants, elderly people, and immunocompromised patients can cause severe disease, thus calling for the development of effective and safe therapeutics to treat it. Here we reported the design, synthesis and characterization of two peptide-based membrane fusion inhibitors targeting HCoV-229E spike protein heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains, 229E-HR1P and 229E-HR2P, respectively. We found that 229E-HR1P and 229E-HR2P could interact to form a stable six-helix bundle and inhibit HCoV-229E spike protein-mediated cell-cell fusion with IC50 of 5.7 and 0.3 µM, respectively. 229E-HR2P effectively inhibited pseudotyped and live HCoV-229E infection with IC50 of 0.5 and 1.7 µM, respectively. In a mouse model, 229E-HR2P administered intranasally could widely distribute in the upper and lower respiratory tracts and maintain its fusion-inhibitory activity. Therefore, 229E-HR2P is a promising candidate for further development as an antiviral agent for the treatment and prevention of HCoV-229E infection.
Publication
Journal: Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]
August/16/2011
Abstract
Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.
Publication
Journal: Viruses
September/5/2012
Abstract
Acute respiratory illnesses (ARIs) with unconfirmed infectious aetiologies peak at different times of the year. Molecular diagnostic assays reduce the number of unconfirmed ARIs compared to serology- or culture-based techniques. Screening of 888 inpatient and outpatient respiratory specimens spanning late autumn through to early spring, 2004, identified the presence of a human coronavirus (HCoV) on 74 occasions (8.3% of all specimens and 26.3% of all respiratory virus detections). Prevalence peaked in August (late winter in the southern hemisphere) when they were detected in 21.9% of specimens tested. HCoV-HKU1 and HCoV-OC43 comprised 82.4% of all HCoVs detected. Positive specimens were used to develop novel reverse transcriptase real-time PCRs (RT-rtPCRs) for HCoV detection. An objective clinical severity score was assigned to each positive HCoV patient. Severity scores were similar to those from a random selection of young children who were positive for respiratory syncytial virus at a different time but from the same specimen population. During the cooler months of 2004, sensitive and specific RT-rtPCRs identified the concurrent circulation of all four HCoVs, a quarter of which co-occurred with another virus and most of which were from children under the age of two years.
Publication
Journal: Journal of Clinical Microbiology
February/2/2005
Abstract
Using paired serum samples obtained from patients with illness associated with increases in anti-human coronavirus OC43 (HCoV-OC43) or anti-HCoV-229E antibodies, we examined the possibility of false-positive results detected in a recombinant severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein immunoglobulin G enzyme-linked immunosorbent assay (ELISA). Three of the 21 and 1 of the 7 convalescent-phase serum samples from persons with increases in antibodies against HCoV-OC43 and HCoV-229E, respectively, tested positive by the recombinant SARS-CoV nucleocapsid protein-based ELISA. None of these samples were found to contain a specific antibody in the recombinant SARS-CoV spike polypeptide-based Western blot assay.