ORIGINAL ARTICLE 10.1111/J.1469-0691.2006.01506.X
Total Page：16
File Type：pdf, Size：1020Kb
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ORIGINAL ARTICLE 10.1111/j.1469-0691.2006.01506.x Detection of human coronavirus NL63, human metapneumovirus and respiratory syncytial virus in children with respiratory tract infections in south-west Sweden A. Koetz1, P. Nilsson1, M. Linde´n2, L. van der Hoek3 and T. Ripa1 1Department of Clinical Microbiology and Infection Control, 2Pediatric Department, Hospital of Halmstad, Halmstad, Sweden and 3Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands ABSTRACT Two recently detected viruses, human metapneumovirus (hMPV) and coronavirus NL63 (HCoV-NL63), have been associated with acute respiratory tract infections, particularly in young children. This study investigated the frequency of hMPV and HCoV-NL63 infections in Swedish children by screening 221 nasopharyngeal aspirates, collected between November 2003 and May 2005, from 212 children attending the paediatric department of a county hospital in Sweden or submitted from local general practitioners. The samples were originally submitted to be tested for respiratory syncytial virus (RSV), and were examined retrospectively for hMPV and HCoV-NL63 by RT-PCR. Of the 212 patients, 101 were positive for RSV (48%), 22 (10%) were positive for hMPV, and 12 (6%) were positive for HCoV-NL63. The frequency of HCoV-NL63 infection increased from 1% in 2003–2004 to 10% in 2004–2005. Sequence analysis of parts of the coronavirus genomes showed considerable similarity to the HCoV-NL63 prototype sequence. The study demonstrated that HCoV-NL63 and hMPV occur in south-west Sweden with essentially the same frequency, seasonal distribution and clinical characteristics as have been reported in other countries. Keywords Bronchiolitis, children, human coronavirus NL63, human metapneumovirus, respiratory syncytial virus Original Submission: 30 January 2005; Revised Submission: 13 February 2006; Accepted: 12 March 2006 Clin Microbiol Infect 2006; 12: 1089–1096 symptoms and respiratory tract diseases similar INTRODUCTION to those caused by RSV, mainly in infants and Respiratory syncytial virus (RSV) is the most elderly individuals. The epidemiology of hMPV common viral cause of lower respiratory tract infections appears to be seasonal, with a peak in infection in young children, followed by para- late winter and early spring. In the case of influenza virus, influenza virus and other viruses, HCoV-NL63, there have been a number of but no aetiological cause is identified for many worldwide reports that describe an association cases of infection [1–5]. The number of unidenti- with both upper and lower respiratory tract fied causes has decreased following the discovery infections, and specifically with clinical croup of human metapneumovirus (hMPV) in 2001 [6], [18–26]. and may be reduced still further following the The present study presents data obtained with discovery of human coronavirus NL63 (HCoV- 221 nasopharyngeal aspirates collected between NL63) in 2004 [7,8]. November 2003 and May 2005 from 212 children Infections with hMPV have been reported in with symptoms of upper and lower respiratory many countries, suggesting a worldwide distri- tract infections in Halmstad, Sweden. The sam- bution [9–17]. hMPV seems to cause a range of ples were analysed for the presence of RSV, hMPV and HCoV-NL63. To simplify the diag- nostic procedures on bronchiolitis samples, the Corresponding author and reprint requests: A. Koetz, Depart- RSV, hMPV and HCoV-NL63 real-time RT-PCRs ment of Clinical Microbiology and Infection Control, Hospital of Halmstad, 30185 Halmstad, Sweden were performed simultaneously on the same E-mail: [email protected] platform. Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases 1090 Clinical Microbiology and Infection, Volume 12 Number 11, November 2006 MATERIALS AND METHODS primer (Table S1) at 1 ng ⁄ lL. Nested PCRs were performed with 2.5 lL of the product from the first-round PCRs. Final Clinical specimens and patient data concentrations in the 50-lL reaction volumes were AmpliTaq- Between 1 November 2003 and 31 May 2005, nasopharyngeal Gold 0.02 U ⁄ lL, 100 lM dNTPs, 20 mM Tris-HCL, pH 8.3, ⁄ l aspirates submitted for RSV testing were examined consecu- bovine serum albumin 100 ng L, 2 mM MgCl2, 50 mM KCl tively. RNA was separated from these samples (see below) and and each primer at 1 ng ⁄ lL (Table S1). stored immediately at )70°C. More than 95% of the specimens Thermocycling conditions for the replicase gene PCR and originated from the outpatient clinic and inpatient wards of the N gene PCR were identical. The first- and the second- the Paediatric Department of Halmstad Hospital, and the round PCRs differed only in the number of cycles, and remainder (<5%) from general practitioners in the county of comprised 95°C for 5 min, followed by 35 cycles (first-round Halland, Sweden. All specimens were obtained from patients PCR) or 25 cycles (second-round PCR) of 95°C for 1 min, 55°C aged <10 years with symptoms of respiratory tract infection. for 1 min and 72°C for 2 min, and finally 72°C for 10 min. PCR Clinical and demographical patient data were obtained from products were separated by electrophoresis on agarose gels the medical records of the patients and recorded on a standard and visualised by staining with ethidium bromide. Positive form. Fig. 1 shows the age distribution of the patients; >90% of and negative controls were used in each RT-PCR and in each the samples were taken from patients aged <1 year. PCR. The positive control consisted of RNA isolated from passage of HCoV-NL63 in LLC-MK2 cells [7], using an amount of RNA equivalent to 105)106 RNA copies for the RT-PCR and RNA extraction subsequent PCRs; the negative control was sterile water. RNA was extracted from c. 140 lL of each nasopharyngeal aspirate with a QiaAmp Viral RNA Mini Kit (Qiagen, Hilden, Real-time RT-PCR assay for HCoV-NL63 Germany) in accordance with the manufacturer’s instructions. RNA extraction and storage at )70°C were completed on the A 98-bp segment of the N gene was amplified using a one-step day that the sample arrived at the laboratory. The dates of real-time RT-PCR and primers, as described by Fouchier et al. sample collection and arrival at the laboratory were identical [8]. Briefly, the final volume of 25 lL contained 3 lL of RNA for 80% of the samples, while the remaining 20% had been preparation, 800 nM each primer, 300 nM probe, 1· TaqMan stored at 4°C overnight and were delivered the following day. Universal PCR Master Mix and 1· MultiScribe and RNAse Inhibitor (TaqMan One-Step RT-PCR Master Mix Reagents; Roche, Branchburg, NJ, USA). Cycling parameters comprised Conventional RT-PCR and sequencing for HCoV-NL63 30 min at 48°C, 10 min at 95°C, and 45 cycles of 15 s at 95°C To detect HCoV-NL63 RNA, two separate nested PCRs were and 1 min at 60°C. Reverse transcription of RNA, amplification performed, following one combined RT step. The PCRs and detection of cDNA were performed using an ABI Prism amplified sequences in the replicase and N genes [7]. Reverse 7000 TaqMan instrument (Applied Biosystems, Foster City, transcription was performed using 20 lL of each of the RNA CA, USA). The positive control was the same material used for preparations. The 40-lL RT-PCR mixes contained MMLV- the conventional PCR, but at a concentration of 30–300 RNA reverse transcriptase (Invitrogen, Carlsbad, CA, USA) 5 U ⁄ lL, copies ⁄ PCR. Part of the 5¢-region of the spike insert gene was 500 lM dNTPs, 10 mM Tris-HCl, pH 8.3, Triton-X 0.1% v ⁄ v, sequenced if a PCR product for this gene was generated with 5.5 mM MgCl2, 50 mM KCl and the two RT primers repSZ-RT the primers listed in Table S1; if not, the PCR product from the and N3-RT (Table S1; see Supplementary material) at real-time RT-PCR assay for HCoV-NL63 was used as the 250 pg ⁄ lL. The reaction mix was incubated at 37°C for 90 min. template for sequencing. The PCR products were sequenced In the first-round PCR, 20 lL of the cDNA was added to directly using a BigDye Terminator v.3.1 Cycle Sequencing Kit final reaction volumes of 50 lL containing AmpliTaqGold (Applied Biosystems) with an ABI 3100 Genetic Analyser 0.025 U ⁄ lL, 50 lM dNTPs, 12 mM Tris-HCl, pH 8.3, bovine (Applied Biosystems) at the BioMedical Laboratory, Furulund, ⁄ l serum albumin 60 ng L, 1 mM MgCl2, 30 mM KCl and each Sweden. Both sense and anti-sense strands of the PCR products were sequenced. 50 45 Real-time RT-PCR assay for hMPV 40 A segment of the F gene was amplified with a one-step real-time 35 RT-PCR based on the method described by Maertzdorf et al. [27], which detects all known hMPV genotypes. In brief, the final 30 25-lL volume contained 3 lL of RNA preparation, 300 nM both 25 primers (Table S1), 200 nM probe, 1· TaqMan Universal PCR · 20 Master Mix and 1 MultiScribe and RNAse Inhibitor. As this assay was performed in combination with the HCoV-NL63 and 15 the RSV assays, the cycling parameters and the instrument were Number of patients 10 the same as those described for HCoV-NL63. 5 The positive control consisted of RNA transcribed in vitro 0 with nucleotide sequences derived from an hMPV isolate of <1 1 2 3 4 5 6 7 8 9 10 11 12 13- >24 lineage A (kindly provided by L. P. Nielsen, University Age (months) 24 Hospital, Odense, Denmark).