Identification of a new human coronavirus methods
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Detection of HCoV-NL63 in patient specimens
Having confirmed that the cultured coronavirus originated from the child, the question remained as to whether this was an isolated clinical case, or whether HCoV-NL63 is circulating in humans. To address this question, we used two diagnostic RT-PCR assays to examine respiratory specimens of hospitalized individuals and those visiting the outpatient clinic between December 2002 and August 2003 ( ). We identified seven additional individuals carrying HCoV-NL63 ( ). Sequence analysis of the PCR products indicated the presence of a few characteristic point mutations in several samples, suggesting that several viruses with different molecular markers may be cocirculating ( and online). At least five of the HCoV-NL63-positive individuals suffered from respiratory tract illness; the clinical data of two individuals was not available. Including the index case, five of the patients were childr...
Methods
The virus was cultured on LLC-MK2 cells. Details of virus culture and patient descriptions are available in online. To remove residual cells and mitochondria, 110 µl of virus culture supernatant was spun for 10 min at maximum speed (13,500 r.p.m.) in an Eppendorf microcentrifuge. To remove chromosomal DNA and mitochondrial DNA from the lysed cells, 100 µl of supernatant was transferred to a fresh tube and treated with DNase I for 45 min at 37 °C (Ambion). Nucleic acids were extracted as described. A reverse transcription reaction was performed with random hexamer primers (Amersham Bioscience) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT; Invitrogen). Second-strand DNA synthesis was carried out with Sequenase II (Amersham Bioscience), without further addition of a primer. A phenol-chloroform extraction was followed by ethanol precipitation.
Methods
cDNA-AFLP was performed essentially as described, with some modifications. The double-stranded DNA was digested with the Hin P1I and Mse I restriction enzymes (New England Biolabs). Mse I and Hin P1I anchors (see below) were subsequently added, along with 5U ligase enzyme (Invitrogen) in the supplied ligase buffer, for 2 h at 37 °C. The Mse I and Hin P1I anchors were prepared by mixing a top-strand oligonucleotide (5′-CTCGTAGACTGCGTACC-3′ for the Mse I anchor and 5′-GACGATGAGTCCTGAC-3′ for the Hin P1I anchor) with a bottom-strand oligonucleotide (5′-TAGGTACGCAGTC-3′ for the Mse I anchor and 5′-CGGTCAGGACTCAT-3′ for the Hin P1I anchor) in a 1:40 dilution of ligase buffer. Twenty cycles of PCR were carried out with 10 µl of the ligation mixture, 100 ng of Hin P1I standard primer (5′-GACGATGAGTCCTGACCGC-3′) and 100 ng...
Diagnostic RT-PCR.
A total of 614 respiratory samples were collected from 493 individuals between December 2002 and August 2003 at the Academic Medical Center in Amsterdam. The specimens included oral and nasopharyngeal aspirates, throat swabs, bronchoalveolar lavage and sputum. The samples had been collected for routine viral diagnostic screening of people suffering from upper and/or lower respiratory tract diseases, and the patients consented that their samples be used for testing of respiratory viruses that included coronaviruses. We used 100 µl of each sample in a Boom extraction. The diagnostic assay was designed based on the sequence of the 1b gene. The reverse transcription was performed with MMLV-RT (Invitrogen), using 10 ng of reverse transcription primer (repSZ-RT, 5′-CCACTATAAC-3′; coordinate 16232 in HCoV-NL63). The entire reverse transcription mixture was added to the firs...
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Evidence quotes (4)
Having confirmed that the cultured coronavirus originated from the child, the question remained as to whether this was an isolated clinical case, or whether HCoV-NL63 is circulating in humans. To address this question, we used two diagnostic RT-PCR assays to examine respiratory specimens of hospitalized individuals and those visiting the outpatient clinic between December 2002 and August 2003 ( ). We identified seven additional individuals carrying HCoV-NL63 ( ). Sequence analysis of the PCR products indicated the presence of a few characteristic point mutations in several samples, suggesting that several viruses with different molecular markers may be cocirculating ( and online). At least five of the HCoV-NL63-positive individuals suffered from respiratory tract illness; the clinical data of two individuals was not available. Including the index case, five of the patients were children less than 1 year old, and three patients were adults. Two adults were likely to be immunosuppressed, as one of them was a bone marrow transplant recipient and the other an HIV-positive patient suffering from AIDS, with very low CD4 + cell counts ( ). No clinical data was available for the third a...
The virus was cultured on LLC-MK2 cells. Details of virus culture and patient descriptions are available in online. To remove residual cells and mitochondria, 110 µl of virus culture supernatant was spun for 10 min at maximum speed (13,500 r.p.m.) in an Eppendorf microcentrifuge. To remove chromosomal DNA and mitochondrial DNA from the lysed cells, 100 µl of supernatant was transferred to a fresh tube and treated with DNase I for 45 min at 37 °C (Ambion). Nucleic acids were extracted as described. A reverse transcription reaction was performed with random hexamer primers (Amersham Bioscience) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT; Invitrogen). Second-strand DNA synthesis was carried out with Sequenase II (Amersham Bioscience), without further addition of a primer. A phenol-chloroform extraction was followed by ethanol precipitation.
cDNA-AFLP was performed essentially as described, with some modifications. The double-stranded DNA was digested with the Hin P1I and Mse I restriction enzymes (New England Biolabs). Mse I and Hin P1I anchors (see below) were subsequently added, along with 5U ligase enzyme (Invitrogen) in the supplied ligase buffer, for 2 h at 37 °C. The Mse I and Hin P1I anchors were prepared by mixing a top-strand oligonucleotide (5′-CTCGTAGACTGCGTACC-3′ for the Mse I anchor and 5′-GACGATGAGTCCTGAC-3′ for the Hin P1I anchor) with a bottom-strand oligonucleotide (5′-TAGGTACGCAGTC-3′ for the Mse I anchor and 5′-CGGTCAGGACTCAT-3′ for the Hin P1I anchor) in a 1:40 dilution of ligase buffer. Twenty cycles of PCR were carried out with 10 µl of the ligation mixture, 100 ng of Hin P1I standard primer (5′-GACGATGAGTCCTGACCGC-3′) and 100 ng of Mse I standard primer (5′-CTCGTAGACTGCGTACCTAA-3′). Five microliters of this PCR product was used as input in the second 'selective' amplification step, along with 100 ng Hin P1I N-primer and 100 ng Mse I N-primer (the 'N' indicates that the standard primers were extended with one nu...
A total of 614 respiratory samples were collected from 493 individuals between December 2002 and August 2003 at the Academic Medical Center in Amsterdam. The specimens included oral and nasopharyngeal aspirates, throat swabs, bronchoalveolar lavage and sputum. The samples had been collected for routine viral diagnostic screening of people suffering from upper and/or lower respiratory tract diseases, and the patients consented that their samples be used for testing of respiratory viruses that included coronaviruses. We used 100 µl of each sample in a Boom extraction. The diagnostic assay was designed based on the sequence of the 1b gene. The reverse transcription was performed with MMLV-RT (Invitrogen), using 10 ng of reverse transcription primer (repSZ-RT, 5′-CCACTATAAC-3′; coordinate 16232 in HCoV-NL63). The entire reverse transcription mixture was added to the first PCR mixture containing 100 ng of primer repSZ-1 (5′-GTGATGCATATGCTAATTTG-3′; coordinate 15973) and 100 ng of primer repSZ-3 (5′-CTCTTGCAGGTATAATCCTA-3′; coordinate 16210). The PCR reaction consisted of the following steps: 95 °C for 5 min; then 35 cycles of 95 °C...
Machine-readable layer
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