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.

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.