The Experts below are selected from a list of 1245 Experts worldwide ranked by ideXlab platform
Lia Van Der Hoek - One of the best experts on this subject based on the ideXlab platform.
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replication dependent downregulation of cellular angiotensin converting enzyme 2 protein expression by Human Coronavirus NL63
Journal of General Virology, 2012Co-Authors: Ronald Dijkman, Krzysztof Pyrc, Maarten F Jebbink, Aleksandra Milewska, Martin Deijs, Elena Buelow, Anna Van Der Bijl, Lia Van Der HoekAbstract:Like severe acute respiratory syndrome Coronavirus (SARS-CoV), Human Coronavirus (HCoV)-NL63 employs angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry. SARS-CoV infection causes robust downregulation of cellular ACE2 expression levels and it has been suggested that the SARS-CoV effect on ACE2 is involved in the severity of disease. We investigated whether cellular ACE2 downregulation occurs at optimal replication conditions of HCoV-NL63 infection. The expression of the homologue of ACE2, the ACE protein not used as a receptor by HCoV-NL63, was measured as a control. A specific decrease for ACE2 protein level was observed when HCoV-NL63 was cultured at 34 °C. Culturing the virus at the suboptimal temperature of 37 °C resulted in low replication of the virus and the effect on ACE2 expression was lost. We conclude that the decline of ACE2 expression is dependent on the efficiency of HCoV-NL63 replication, and that HCoV-NL63 and SARS-CoV both affect cellular ACE2 expression during infection.
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simultaneous treatment of Human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome Coronavirus entry
Journal of Virology, 2012Co-Authors: Miyuki Kawase, Lia Van Der Hoek, Fumihiro Taguchi, Kazuya Shirato, Shutoku MatsuyamaAbstract:ABSTRACT The type II transmembrane protease TMPRSS2 activates the spike (S) protein of severe acute respiratory syndrome Coronavirus (SARS-CoV) on the cell surface following receptor binding during viral entry into cells. In the absence of TMPRSS2, SARS-CoV achieves cell entry via an endosomal pathway in which cathepsin L may play an important role, i.e., the activation of spike protein fusogenicity. This study shows that a commercial serine protease inhibitor (camostat) partially blocked infection by SARS-CoV and Human Coronavirus NL63 (HCoV-NL63) in HeLa cells expressing the receptor angiotensin-converting enzyme 2 (ACE2) and TMPRSS2. Simultaneous treatment of the cells with camostat and EST [(23,25) trans -epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester], a cathepsin inhibitor, efficiently prevented both cell entry and the multistep growth of SARS-CoV in Human Calu-3 airway epithelial cells. This efficient inhibition could be attributed to the dual blockade of entry from the cell surface and through the endosomal pathway. These observations suggest camostat as a candidate antiviral drug to prevent or depress TMPRSS2-dependent infection by SARS-CoV.
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burden of disease due to Human Coronavirus NL63 infections and periodicity of infection
Journal of Clinical Virology, 2010Co-Authors: Lia Van Der Hoek, Ben Berkhout, Michel De Vries, Ronald Dijkman, Astrid Vabret, Gabriele Ihorst, Klaus Sure, Johannes Forster, Klaus UberlaAbstract:Abstract Background The disease burden caused by recently identified respiratory viruses like HCoV-NL63 is unknown. Objectives We determined the burden of disease due to HCoV-NL63 infections using the population-based PRI.DE cohort of children under the age of 3 with lower respiratory tract infections (LRTIs). Study design In total 1756 respiratory samples, from hospitalized children or children who visited the outpatient clinic, were tested for HCoV-NL63. Sampling covered a period of 2 years and the frequency of infection in different years was compared to other Western European studies that tested for this virus in 2 or more consecutive years. Results Sixty-nine samples were HCoV-NL63 positive, 35 were with high loads, and of these 25 were single HCoV-NL63 infections. Based on the number of children with high HCoV-NL63 infection and no additional infection, the overall annual incidence in outpatients was 7 per 1000 children per year (95% confidence interval (CI) 3–13 per 1000 children per year), which can be extrapolated to an absolute number of 16,929 visits to the physician due to an HCoV-NL63 infection in Germany per year. The estimated hospitalization rate is 22 per 100,000 children (95% CI: 7–49 per 100,000 children per year). This number reflects 522 HCoV-NL63 children in Germany per year. A large year-to-year difference in HCoV-NL63 infection frequency was observed. Combining these data with those of other studies in Western Europe revealed that HCoV-NL63 infections follow a 2-year inter-epidemic period with peaks of infection in the winters of 2000/2001, 2002/2003 and 2004/2005 (p Conclusions HCoV-NL63 infection in children below 3 years of age often requires a visit to the physician in an outpatient clinic, especially during peak-years, but hospitalizations are relatively infrequent.
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Human Coronavirus NL63 and 229e seroconversion in children
Journal of Clinical Microbiology, 2008Co-Authors: Ronald Dijkman, Krzysztof Pyrc, Marcel A. Müller, Maarten F Jebbink, Nawal Bahia El Idrissi, Taco W Kuijpers, Hans L Zaaijer, Lia Van Der HoekAbstract:In 2004, the novel respiratory Human Coronavirus NL63 (HCoV-NL63) was identified, and subsequent research revealed that the virus has spread worldwide. HCoV-229E is a close relative of HCoV-NL63, and infection with either virus can lead to the hospitalization of young children, immunocompromised persons, and the elderly. Children infected with HCoV-NL63 often develop croup, with obstruction of the airway. In this study we investigated at which age children are confronted for the first time with an HCoV-NL63 infection and, thus, at which age they seroconvert to HCoV-NL63 positivity. We designed a recombinant HCoV-229E and a recombinant HCoV-NL63 nucleocapsid protein enzyme-linked immunosorbent assay and performed a seroepidemiology survey on longitudinal and cross-sectional serum samples. The longitudinal serum samples were collected from 13 newborns, and data for those newborns were available from multiple time points spanning a period of at least 18 months. For the cross-sectional survey we tested serum samples of 139 children, including newborns to children 16 years of age. In examinations of the longitudinal serum samples we observed that all of the children had maternal anti-NL63 and anti-229E antibodies at birth that disappeared within 3 months. Seven of the 13 children became HCoV-NL63 seropositive during follow-up, whereas only 2 became HCoV-229E seropositive. The serology data of the cross-sectional serum samples revealed that 75% and 65% of the children in the age group 2.5 to 3.5 years were HCoV-NL63 and HCoV-229E seropositive, respectively. We conclude that on average, HCoV-NL63 and HCoV-229E seroconversion occurs before children reach the age of 3.5 years.
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mosaic structure of Human Coronavirus NL63 one thousand years of evolution
Journal of Molecular Biology, 2006Co-Authors: Krzysztof Pyrc, Ben Berkhout, Maarten F Jebbink, Ronald Dijkman, Lea Deng, Howard A Ross, Lia Van Der HoekAbstract:Before the SARS outbreak only two Human Coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth Human Coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib Coronaviruses to be approximately 3 x 10(-4) substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.
Krzysztof Pyrc - One of the best experts on this subject based on the ideXlab platform.
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membrane protein of Human Coronavirus NL63 is responsible for interaction with the adhesion receptor
Journal of Virology, 2019Co-Authors: Antonina Naskalska, Aleksandra Milewska, Artur Szczepanski, Agnieszka Dabrowska, Krzysztof Jasik, Krzysztof PyrcAbstract:Human Coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation.IMPORTANCE It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we show that the M protein is also an important player during early stages of HCoV-NL63 infection and that the concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented here may also be important for future research into vaccine or drug development.
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Structural Characterization of Human Coronavirus NL63 N Protein.
Journal of Virology, 2017Co-Authors: Bozena Szelazek, Wojciech Kabala, Michał Burmistrz, Michal Zdzalik, Dominik Florek, Krzysztof Kus, Aleksandra Twarda-clapa, Przemyslaw Golik, Benedykt Wladyka, Krzysztof PyrcAbstract:ABSTRACT Coronaviruses are responsible for upper and lower respiratory tract infections in Humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with Human Coronavirus NL63 (HCoV-NL63), an alphaCoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphaCoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-A resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections. IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in Humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of Coronavirus physiology and may facilitate future efforts to control the associated infections.
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Human Coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells
Journal of Virology, 2014Co-Authors: Aleksandra Milewska, Paulina Nowak, Jan Potempa, Miroslaw Zarebski, Karol Stozek, Krzysztof PyrcAbstract:Human Coronavirus NL63 (HCoV-NL63) is an alphaCoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63. IMPORTANCE ACE2 protein was proposed as a receptor for HCoV-NL63 already in 2005, but an in-depth analysis of early events during virus infection had not been performed thus far. Here, we show that the ACE2 protein is required for viral entry but that it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating the interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and that a newly described interaction may be essential for understanding the infection process and, possibly, also assist in drug design.
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stability of infectious Human Coronavirus NL63
Journal of Virological Methods, 2014Co-Authors: Dominik Florek, Michał Burmistrz, Jan Potempa, Krzysztof PyrcAbstract:The Human Coronavirus NL63 was identified in 2004 and subsequent studies showed its worldwide distribution. Infection with this pathogen is associated with upper and lower respiratory tract diseases of mild to moderate severity. Furthermore, HCoV-NL63 is the main cause of croup in children. Within this study an optimal protocol for freeze-drying that allows safe and effective preservation of HCoV-NL63 infectious material was developed. Lyophilized virus preparations can be stored either at ambient temperature or at +4°C. In the latter case samples may be stored for at least two months. Surprisingly, conducted analysis showed that HCoV-NL63 virions are exquisitely stable in liquid media and can be stored also without preservatives at ambient temperature for up to 14 days.
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replication dependent downregulation of cellular angiotensin converting enzyme 2 protein expression by Human Coronavirus NL63
Journal of General Virology, 2012Co-Authors: Ronald Dijkman, Krzysztof Pyrc, Maarten F Jebbink, Aleksandra Milewska, Martin Deijs, Elena Buelow, Anna Van Der Bijl, Lia Van Der HoekAbstract:Like severe acute respiratory syndrome Coronavirus (SARS-CoV), Human Coronavirus (HCoV)-NL63 employs angiotensin-converting enzyme 2 (ACE2) as a receptor for cellular entry. SARS-CoV infection causes robust downregulation of cellular ACE2 expression levels and it has been suggested that the SARS-CoV effect on ACE2 is involved in the severity of disease. We investigated whether cellular ACE2 downregulation occurs at optimal replication conditions of HCoV-NL63 infection. The expression of the homologue of ACE2, the ACE protein not used as a receptor by HCoV-NL63, was measured as a control. A specific decrease for ACE2 protein level was observed when HCoV-NL63 was cultured at 34 °C. Culturing the virus at the suboptimal temperature of 37 °C resulted in low replication of the virus and the effect on ACE2 expression was lost. We conclude that the decline of ACE2 expression is dependent on the efficiency of HCoV-NL63 replication, and that HCoV-NL63 and SARS-CoV both affect cellular ACE2 expression during infection.
Ben Berkhout - One of the best experts on this subject based on the ideXlab platform.
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burden of disease due to Human Coronavirus NL63 infections and periodicity of infection
Journal of Clinical Virology, 2010Co-Authors: Lia Van Der Hoek, Ben Berkhout, Michel De Vries, Ronald Dijkman, Astrid Vabret, Gabriele Ihorst, Klaus Sure, Johannes Forster, Klaus UberlaAbstract:Abstract Background The disease burden caused by recently identified respiratory viruses like HCoV-NL63 is unknown. Objectives We determined the burden of disease due to HCoV-NL63 infections using the population-based PRI.DE cohort of children under the age of 3 with lower respiratory tract infections (LRTIs). Study design In total 1756 respiratory samples, from hospitalized children or children who visited the outpatient clinic, were tested for HCoV-NL63. Sampling covered a period of 2 years and the frequency of infection in different years was compared to other Western European studies that tested for this virus in 2 or more consecutive years. Results Sixty-nine samples were HCoV-NL63 positive, 35 were with high loads, and of these 25 were single HCoV-NL63 infections. Based on the number of children with high HCoV-NL63 infection and no additional infection, the overall annual incidence in outpatients was 7 per 1000 children per year (95% confidence interval (CI) 3–13 per 1000 children per year), which can be extrapolated to an absolute number of 16,929 visits to the physician due to an HCoV-NL63 infection in Germany per year. The estimated hospitalization rate is 22 per 100,000 children (95% CI: 7–49 per 100,000 children per year). This number reflects 522 HCoV-NL63 children in Germany per year. A large year-to-year difference in HCoV-NL63 infection frequency was observed. Combining these data with those of other studies in Western Europe revealed that HCoV-NL63 infections follow a 2-year inter-epidemic period with peaks of infection in the winters of 2000/2001, 2002/2003 and 2004/2005 (p Conclusions HCoV-NL63 infection in children below 3 years of age often requires a visit to the physician in an outpatient clinic, especially during peak-years, but hospitalizations are relatively infrequent.
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mosaic structure of Human Coronavirus NL63 one thousand years of evolution
Journal of Molecular Biology, 2006Co-Authors: Krzysztof Pyrc, Ben Berkhout, Maarten F Jebbink, Ronald Dijkman, Lea Deng, Howard A Ross, Lia Van Der HoekAbstract:Before the SARS outbreak only two Human Coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth Human Coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib Coronaviruses to be approximately 3 x 10(-4) substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.
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core structure of s2 from the Human Coronavirus NL63 spike glycoprotein
Biochemistry, 2006Co-Authors: Qi Zheng, Lia Van Der Hoek, Yiqun Deng, Jie Liu, Ben BerkhoutAbstract:Human Coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS Coronavirus or the prototypic murine Coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an α-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stra...
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Human Coronavirus NL63, a new respiratory virus
Fems Microbiology Reviews, 2006Co-Authors: Lia Van Der Hoek, Krzysztof Pyrc, Ben BerkhoutAbstract:From the mid-1960s onwards, it was believed that only two Human Coronavirus species infect Humans: HCoV-229E and HCoV-OC43. Then, in 2003, a novel member of the Coronavirus family was introduced into the Human population: SARS-CoV, causing an aggressive lung disease. Fortunately, this virus was soon expelled from the Human population, but it quickly became clear that the Human Coronavirus group contains more members then previously assumed, with HCoV-NL63 identified in 2004. Despite its recent discovery, ample results from HCoV-NL63 research have been described. We present an overview of the publications on this novel Coronavirus.
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attachment factor and receptor engagement of sars Coronavirus and Human Coronavirus NL63
Advances in Experimental Medicine and Biology, 2006Co-Authors: Heike Hofmann, Lia Van Der Hoek, Krzysztof Pyrc, Martina Geier, Ben Berkhout, Andrea Marzi, Thomas Gramberg, Stefan PöhlmannAbstract:The cellular membrane constitutes a physical barrier against viral infection. Enveloped viruses developed specialized proteins to overcome this barrier. These proteins, which are often extensively glycosylated, are inserted into the viral membrane and mediate both recognition of target cells and fusion of the viral membrane with a host cell membrane. The latter process allows introduction of the viral genome and associated viral proteins into the host cell lumen and is therefore critical for establishment of productive infection. which in the case of Coronaviruses (CoV) are termed spike (S) proteins, are attractive targets for inhibitors and vaccines. Enveloped viruses evolved two prototypes of glycoproteins to enter target cells, termed class I and class II fusion proteins. 4 Class I fusion proteins are found in, e.g., retroviruses and paramyxoviruses, while, e.g., flaviviruses and alphaviruses encode class II fusion proteins. Both types of fusion proteins exhibit a distinct functional organization, which is reflected by their different spatial orientations. Thus, class I fusion proteins are oriented perpendicular to the cellular membrane and are visible as spikes in electron micrographs, while class II proteins are oriented horizontally relative to the cellular membrane and are well ordered on the virion surface. Viral class I fusion proteins are organized into a globular surface unit (SU), which interacts with cellular receptors, and a transmembrane unit (TM), which harbors highly conserved sequence elements required for membrane fusion. Membrane fusion is initiated by binding of SU to cellular receptor(s) or by exposure of the glycoprotein to low pH, which triggers conformational changes in the glycoprotein that activate TM. TM-driven membrane fusion is initiated by insertion of a N-terminal fusion peptide into the target cell membrane, followed by
Wenjie Tan - One of the best experts on this subject based on the ideXlab platform.
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structure of main protease from Human Coronavirus NL63 insights for wide spectrum anti Coronavirus drug design
Scientific Reports, 2016Co-Authors: Fenghua Wang, Wenjie Tan, Cheng Chen, Kailin Yang, Haitao YangAbstract:First identified in The Netherlands in 2004, Human Coronavirus NL63 (HCoV-NL63) was found to cause worldwide infections. Patients infected by HCoV-NL63 are typically young children with upper and lower respiratory tract infection, presenting with symptoms including croup, bronchiolitis, and pneumonia. Unfortunately, there are currently no effective antiviral therapy to contain HCoV-NL63 infection. CoV genomes encode an integral viral component, main protease (Mpro), which is essential for viral replication through proteolytic processing of RNA replicase machinery. Due to the sequence and structural conservation among all CoVs, Mpro has been recognized as an attractive molecular target for rational anti-CoV drug design. Here we present the crystal structure of HCoV-NL63 Mpro in complex with a Michael acceptor inhibitor N3. Structural analysis, consistent with biochemical inhibition results, reveals the molecular mechanism of enzyme inhibition at the highly conservative substrate-recognition pocket. We show such molecular target remains unchanged across 30 clinical isolates of HCoV-NL63 strains. Through comparative study with Mpros from other Human CoVs (including the deadly SARS-CoV and MERS-CoV) and their related zoonotic CoVs, our structure of HCoV-NL63 Mpro provides critical insight into rational development of wide spectrum antiviral therapeutics to treat infections caused by Human CoVs.
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visual detection of Human Coronavirus NL63 by reverse transcription loop mediated isothermal amplification
Chinese journal of virology, 2016Co-Authors: Heyuan Geng, Shengqiang Wang, Xiaoqian Xie, Yu Xiao, Ting Zhang, Wenjie TanAbstract:A simple and sensitive assay for rapid detection of Human Coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different Human Coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of Human Coronavirus NL63.
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characterization of complete genome sequences of Human Coronavirus NL63 strains derived from chinese patients
Chinese journal of microbiology and immunology, 2014Co-Authors: Heyuan Geng, Lijin Cui, Li Zhao, Wenjie TanAbstract:Objective To sequence and analyze the complete genome of two Human Coronavirus NL63 （HCoV-NL63） strains collected from Beijing Children Hospital .Methods Eighteen pairs of primers were designed according to the gene sequences of HCoV-NL63 reference strain （ HCoV-NL63_Amsterdam 1） and used to amplify the target fragments covering the complete genome of HCoV-NL63 strains.Rapid amplification of cDNA ends （ RACE） and RT-PCR assays were used to amplify the full length genome of HCoV-NL63 strains.Phylogenetic analysis was conducted by using Mega 5.0 software.Results The complete genome sequences of the two HCoV-NL63 strains were 27 538 bp in length, showing a homology of 99.1%in nucleotide sequences .There were 15 consecutive bases deleted from 1a region.The systematic phylogenetic analysis demonstrated that four genotypes of NL 63 virus including A , B, C and D have been identified , and two domestic strains were belonged to the new genotype D .Conclusion The complete genome sequences of two domestic HCoV-NL63 isolates were identified for the first time .This study provided evidence for further investigation on molecular epidemiology of HCoV-NL63 in China . Key words: Complete genome sequence; Phylogenetic analysis
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prokaryotic expression and characterization of two recombinant receptor binding domain rbd proteins of Human Coronavirus NL63 hcov NL63
Chinese journal of virology, 2013Co-Authors: Hui Chang, Weimin Zhou, Min Zhao, Guoxia Zhao, Huijuan Wang, Jimin Gao, Bing Liu, Wenjie TanAbstract:The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.
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characterization and complete genome sequence of Human Coronavirus NL63 isolated in china
Journal of Virology, 2012Co-Authors: Heyuan Geng, Lijin Cui, Zhengde Xie, Li Zhao, Wenjie TanAbstract:ABSTRACT Human Coronavirus NL63 (HCoV-NL63) was first discovered in Amsterdam in 2004 and was identified as a new Human respiratory Coronavirus. We here report the first complete genome sequence of HCoV-NL63 strain CBJ 037 isolated in 2008 from a patient with bronchitis in Beijing, China.
Graham Simmons - One of the best experts on this subject based on the ideXlab platform.
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a 3 4 a cryo electron microscopy structure of the Human Coronavirus spike trimer computationally derived from vitrified NL63 virus particles
QRB discovery, 2020Co-Authors: Kaiming Zhang, Graham Simmons, Jing Jin, Grigore Pintilie, David Chmielewski, Michael F Schmid, Wah ChiuAbstract:Human Coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV-1 and SARS-CoV-2. Here, we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-A resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a three-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other sites, as well as different glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies or drugs, in the native state of the Coronavirus particles.
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a 3 4 a cryo em structure of the Human Coronavirus spike trimer computationally derived from vitrified NL63 virus particles
bioRxiv, 2020Co-Authors: Kaiming Zhang, Graham Simmons, Wah Chiu, Jing Jin, Grigore Pintilie, David Chmielewski, Michael F SchmidAbstract:Human Coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV and SARS-CoV-2. Here we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-A resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a 3-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other amino acid positions as well as differences in glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies, or drugs, in the native state of the Coronavirus particles.
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decontamination of sars cov 2 and other rna viruses from n95 level meltblown polypropylene fabric using heat under different humidities
ACS Nano, 2020Co-Authors: Rafael K Campos, Grace H Rafael, Mervin Zhao, Lei Liao, Graham Simmons, Scott C Weaver, Wah Chiu, Jing Jin, Steven Chu, Yi CuiAbstract:In March of 2020, the World Health Organization declared a pandemic of Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). The pandemic led to a shortage of N95-grade filtering facepiece respirators (FFRs), especially surgical-grade N95 FFRs for protection of healthcare professionals against airborne transmission of SARS-CoV-2. We and others have previously reported promising decontamination methods that may be applied to the recycling and reuse of FFRs. In this study we tested disinfection of three viruses, including SARS-CoV-2, dried on a piece of meltblown fabric, the principal component responsible for filtering of fine particles in N95-level FFRs, under a range of temperatures (60-95 °C) at ambient or 100% relative humidity (RH) in conjunction with filtration efficiency testing. We found that heat treatments of 75 °C for 30 min or 85 °C for 20 min at 100% RH resulted in efficient decontamination from the fabric of SARS-CoV-2, Human Coronavirus NL63 (HCoV-NL63), and another enveloped RNA virus, chikungunya virus vaccine strain 181/25 (CHIKV-181/25), without lowering the meltblown fabric's filtration efficiency.
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decontamination of sars cov 2 and other rna viruses from n95 level meltblown polypropylene fabric using heat under different humidities
medRxiv, 2020Co-Authors: Rafael K Campos, Grace H Rafael, Mervin Zhao, Lei Liao, Graham Simmons, Scott C Weaver, Wah ChiuAbstract:In March of 2020, the World Health Organization declared a pandemic of Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). The pandemic led to a shortage of N95-grade filtering facepiece respirators (FFRs), especially for protection of healthcare professionals against airborne transmission of SARS-CoV-2. We and others have previously reported promising decontamination methods that may be applied to the recycling and reuse of FFRs. In this study we tested disinfection of three viruses including SARS-CoV-2, dried on a piece of meltblown fabric, the principal component responsible for filtering of fine particles in N95-level FFRs, under a range of temperatures (60-95°C) at ambient or 100% relative humidity (RH) in conjunction with filtration efficiency testing. We found that heat treatments of 75°C for 30 min or 85°C for 20 min at 100% RH resulted in efficient decontamination from the fabric of SARS-CoV-2, Human Coronavirus NL63 (HCoV-NL63) and chikungunya virus vaccine strain 181 (CHIKV-181), without lowering the meltblown fabric9s filtration efficiency.
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decontamination of sars cov 2 and other rna viruses from n95 level meltblown polypropylene fabric using heat under different humidities
medRxiv, 2020Co-Authors: Rafael K Campos, Grace H Rafael, Mervin Zhao, Lei Liao, Graham Simmons, Scott C Weaver, Wah Chiu, Jing Jin, Steven Chu, Yi CuiAbstract:ABSTRACT In March of 2020, the World Health Organization declared a pandemic of Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). The pandemic led to a shortage of N95-grade filtering facepiece respirators (FFRs), especially for protection of healthcare professionals against airborne transmission of SARS-CoV-2. We and others have previously reported promising decontamination methods that may be applied to the recycling and reuse of FFRs. In this study we tested disinfection of three viruses including SARS-CoV-2, dried on a piece of meltblown fabric, the principal component responsible for filtering of fine particles in N95-level FFRs, under a range of temperatures (60-95°C) at ambient or 100% relative humidity (RH) in conjunction with filtration efficiency testing. We found that heat treatments of 75°C for 30 min or 85°C for 20 min at 100% RH resulted in efficient decontamination from the fabric of SARS-CoV-2, Human Coronavirus NL63 (HCoV-NL63), and another enveloped RNA virus, chikungunya virus vaccine strain 181 (CHIKV-181), without lowering the meltblown fabric’s filtration efficiency.