Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia

Abstract

Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 x 10(6) copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 x 10(6) copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.

Figures

FIG. 1.

Genome organization of CoV-HKU1. Overall organization of the 29,926-nucleotide CoV-HKU1 genomic RNA. Predicted ORFs 1a and 1b, encoding the nonstructural polyproteins (p28, p65, and nsp1 to -13) and those encoding the hemagglutinin-esterase, spike, envelope, membrane and nucleocapsid structural proteins are indicated. Arrows indicate putative cleavage sites (with the corresponding nucleotide positions) of the replicase polyprotein encoded by ORF 1a and ORF 1b. ATR and PL1pro and PL2pro represent the acidic tandem repeat and the two papain-like proteases, respectively, in nsp1.

FIG. 2.

Phylogenetic analysis of chymotrypsin-like protease (3CLpro), RNA-dependent RNA polymerase (Pol), helicase, hemagglutinin-esterase (HE), spike (S), envelope (E), membrane (M), and nucleocapsid (N) of CoV-HKU1. The trees were constructed by the neighbor-joining method, using Jukes-Cantor correction and bootstrap values calculated from 1,000 trees. Three hundred three, 928, 595, 418, 1356, 75, 225, and 406 amino acid positions in 3CLpro, Pol, helicase, HE, S, E, M and N, respectively, were included in the analysis. The scale bar indicates the estimated number of substitutions per 10 amino acids. HCoV-229E, human coronavirus 229E; PEDV, porcine epidemic diarrhea virus; PTGV, porcine transmissible gastroenteritis virus; CCoV, canine enteric coronavirus; HCoV-NL63, human coronavirus NL63; HCoV-OC43, human coronavirus OC43; MHV, murine hepatitis virus; BCoV, bovine coronavirus; SDAV, rat sialodacryoadenitis coronavirus; ECoV, equine coronavirus NC99; PHEV, porcine hemagglutinating encephalomyelitis virus; IBV, infectious bronchitis virus; SARS-CoV, SARS coronavirus.

FIG. 3.

Arrangements of proteins in replicase polyprotein in HKU1 compared with those in HCoV-OC43, BCoV, and MHV. Alignment of the AC domains of HCoV-OC43, BCoV, and MHV and the AC domains and ATR (underlined) of CoV-HKU1 in the two patients was generated with ClustalX 1.83. AC domain, acidic domain. GenBank accession numbers are as follows: MHV, NC_001846; BCoV, NC_003045; HCoV-OC43, AY585229.

FIG. 4.

Spike protein of CoV-HKU1. The spike protein (1,356 amino acids) of CoV-HKU1 is depicted by the horizontal bar. SS, N terminal signal sequence (amino acid residues 1 to 13); HR1, heptad repeat 1 (amino acid residues 982 to 1083); HR2, heptad repeat 2 (amino acid residues 1250 to 1297); TM, transmembrane domain (amino acid residues 1301 to 1323). Alignment of the N-terminal region important for receptor binding (amino acid residues 1 to 330) and the region upstream of the cleavage site between S1 and S2 of CoV-HKU1 and other group 2 coronaviruses was done with ClustalX 1.83. Residues that match the CoV-HKU1 sequence exactly are boxed. The three conserved regions (sites I, II, and III) for receptor binding in MHV are shaded. The positions of the four conserved amino acids important for receptor binding in MHV are indicated with arrows. GenBank accession numbers were as follows: MHV, P11224; BCoV, NP_150077; HCoV-OC43, NP_937950; SDAV, AAF97738; PHEV, AAL80031; ECoV, AAQ67205.

FIG. 5.

Sequential quantitative RT-PCR for CoV-HKU1 in NPAs and serum IgG titers against N protein of CoV-HKU1.

FIG. 6.

Western blot analysis of purified recombinant CoV-HKU1 N protein antigen. Prominent immunoreactive protein bands of about 53 kDa were visible on the Western blot that used recombinant N protein as the antigen during the second and fourth weeks of the patient's illness (lanes 2 and 3). Only very faint bands were observed for serum samples obtained from the patient during the first week of the illness (lane 1) and two healthy blood donors (lanes 4 and 5).