A novel pancoronavirus RT-PCR assay: frequent detection of human coronavirus NL63 in children hospitalized with respiratory tract infections in Belgium

Abstract

Background: Four human coronaviruses are currently known to infect the respiratory tract: human coronaviruses OC43 (HCoV-OC43) and 229E (HCoV-229E), SARS associated coronavirus (SARS-CoV) and the recently identified human coronavirus NL63 (HCoV-NL63). In this study we explored the incidence of HCoV-NL63 infection in children diagnosed with respiratory tract infections in Belgium.

Methods: Samples from children hospitalized with respiratory diseases during the winter seasons of 2003 and 2004 were evaluated for the presence of HCoV-NL63 using a optimized pancoronavirus RT-PCR assay.

Results: Seven HCoV-NL63 positive samples were identified, six were collected during January/February 2003 and one at the end of February 2004.

Conclusions: Our results support the notation that HCoV-NL63 can cause serious respiratory symptoms in children. Sequence analysis of the S gene showed that our isolates could be classified into two subtypes corresponding to the two prototype HCoV-NL63 sequences isolated in The Netherlands in 1988 and 2003, indicating that these two subtypes may currently be cocirculating.

Figure 1

Detection of HCoV-NL63 and HCoV-OC43 in samples from patients suffering from severe respiratory symptoms. (A) Number of samples tested per month. (B) Patients infected with HCoV-NL63 and HCoV-OC43. A single HCoV-229E positive sample was isolated in April 2003 (not shown).

Figure 2

Selection of primers for the novel pancoronavirus RT-PCR. Shown is the alignment of 14 coronaviral sequences of a conserved region of the polymerase gene. The forward (Cor-FW) and reverse (Cor-RV) primer sequences are shown at the bottom (Y = C/T, W = A/T, V = A/C/G, R = A/G, H = A/T/C, N = A/C/T/G). The coordinates of Cor-FW and Cor-RV are 14017 and 14248, respectively, in the HCoV-NL63 complete genome sequence. The 14 coronavirus sequences used here are available from GenBank under the following accession numbers: HCoV-NL63, AY567487; HCoV-229E, AF304460; infectious bronchitis virus (IBV), Z30541; SARS-CoV, AY313906; HCoV-OC43, AY391777; PEDV, AF353511; bovine coronavirus (BCoV), AF391541; transmissible gastroenteritis virus, AF304460; MHV, X51939; PHEV, AF124988; sialodacryoadenitis virus (SDAV), AF124990; turkey coronavirus (TCoV), AF124991; canine respiratory coronavirus (CRCV), AY150273; feline infectious peritonitis virus (FIPV), AF124987.

Figure 3

Gel electrophoresis after pancoronavirus RT-PCR assay. The indicated band of 251 bp corresponds with the expected amplicon size. As a marker Molecular Weight Marker VI was used (Boehringer Mannheim, Germany).

Figure 4

Phylogenetic analysis of the partial ORF1a nucleotide sequences. Accession numbers: HCoV-NL63, AY567487; HCoV-NL, AY518894; HCoV-229E, AF304460; NL-p466, AY567488; NL-p246, AY567489; NL-p251, AY567490; NL-p496, AY567491; NL-p223; AY567492; NL-p248, AY567493; NL-p72, AY567494; CAN39, AY675541; CAN52, AY675542; CAN57, AY675543; CAN140, AY675544; CAN146, AY675545; CAN214, AY675546; CAN449, AY675547; CAN470, AY675548; CAN483, AY675549; CAN495, AY675550; CAN528, AY675551; CAN531, AY675552; CAN543, AY675553.

Figure 5

Phylogenetic analysis of the partial S gene nucleotide sequences based on an alignment of the Belgian spike sequences, spike sequences from the positive samples identified in The Netherlands, and the prototype HCoV-NL63 sequences available in GenBank. Accession numbers: HCoV-NL63, AY567487; HCoV-NL, AY518894. PEDV was used as an outgroup.