Identification of a new human coronavirus

Abstract

Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.

Conflict of interest statement

L.v.d.H. is listed as the inventor on a European and a US patent application, held by Primagen Holding b.v., entitled “Coronavirus nucleic acid, protein, and methods for the generation of vaccine, medicaments and diagnostics.”

Figures

Figure 1. The VIDISCA method.

(a) Schematic overview of steps in VIDISCA method. (b) Examples of VIDISCA-mediated virus identification. Specimens were analyzed using ethidium bromide–stained agarose (parvovirus B19) or Metaphor agarose (HBV and HIV-1) gel electrophoresis. Lane M, DNA molecular weight markers; −, negative controls; +, VIDISCA PCR products for HBV (amplified with primers HinP1I-T/MseI-T), parvovirus B19 (HinP1I standard primer only) or HIV-1 (EcoRI-A/MseI-C primers). (c) VIDISCA PCR products for HCoV-NL63. HinP1I-G and MseI-A primers were used for selective amplification; products were visualized by Metaphor agarose gel electrophoresis. Lanes 1 and 2, duplicate PCR product of cultured HCoV-NL63 harvested from LLC-MK2 cells; 3 and 4, duplicate control supernatant from uninfected LLC-MK2 cells; 5 and 6, duplicate negative controls containing water; M, 25-bp molecular weight marker. Arrow indicates HCoV-NL63 fragment that was excised from gel and sequenced.

Figure 2. Detection of HCoV-NL63 in winter months of 2002 and 2003.

(a) Number of patients tested per month. (b) Percentage of patients positive for HCoV-NL63.

Figure 3. Phylogenetic analysis of RT-PCR sequences of the 1a gene from HCoV-NL63-positive patients.

HCoV-229E was used to root the tree.

Figure 4. HCoV-NL63 genome organization and phylogenetic analysis.

(a) ORFs encoding 1a, 1b, S, ORF3, E, M and N proteins are flanked by 286-nucleotide 5′ UTR and 287-nucleotide 3′ UTR. Coordinates of each ORF are provided in Supplementary Table 1 online. (b) Phylogenetic analysis of HCoV-NL63, using nucleotide sequences predicted to encode 1a, 1b, S, M and N proteins (see Supplementary Methods online for GenBank accession numbers). Red, group 1 viruses; blue, group 2; green, group 3; purple, SARS-CoV. MHV, mouse hepatitis virus; IBV, avian infectious bronchitis virus; BCoV, bovine coronavirus; FCoV, feline enteric coronavirus; CCoV, canine coronavirus; FIPV, feline infectious peritonitis virus; EqCoV, equine coronavirus; TCoV, turkey coronavirus.