tify novel agents. Of the 3 novel agents identifie in the last 3 years-including human metapneumovirus [3] , severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) [4] , and human coronavirus NL63 (HCoV-NL63) [5, 6 ]-2 are coronaviruses. On the basis of serological and phylogenetic characterization, coronaviruses were divided into 3 distinct groups: human coronavirus 229E (HCoV-229E) and HCoV-NL63 are group 1 coronaviruses, and human coronavirus OC43 (HCoV-OC43) is a group 2 coronavirus [7] . HCoV-229E and HCoV-OC43 account for 5%-30% of human respiratory tract infections [8] , whereas, in a few recent studies, HCoV-NL63 was found to be present in 2%-3.6% of respiratory specimens [9] [10] [11] . In 2002 and 2003, the epidemic caused by SARS-CoV affected 18000 people, with 750 deaths [4, [12] [13] [14] [15] [16] [17] [18] . Table 1 . Primers used for amplification and sequencing of the pol, spike (S), and nucleocapsid (N) genes.
Forward primer Reverse primer pol LPW1465 5 -GTTCAAGTGTCGCTGTTCA-3  LPW1822 5 -CTATCATTATCACAATCCACAG-3  LPW1467 5 -GGGTATGAAGTATCATCCTA-3  LPW1825 5 -GATAATCCCAACCCATAAGAAC-3  LPW1826 5 -CATCTTATAAAGGATGTTGAC-3  LPW1829 5 -ACAAACAACACATGCACCTACAC-3  S  LPW1830 5 -TTGCCTATTATTTTACAAGGT-3  LPW1864 5 -ACACTACCTATAACTATAGTAC-3  LPW1832 5 -TATGTTAATAAWACTTTGTATAGTG-3 LPW1866 5 -TACAATTGACAAGAACTAGAAG-3 LPW1836 5 -GATTTGCARTTGGGCAGTTCTGG-3 LPW1868 5 -CCATTAGAATCATACAAAAGAT-3 LPW1840 5 -GGTATTTTTAAAGAAGTTTCTGC-3 LPW1870 5 -AGCTTCAACAAAACCWACATCTG-3 LPW1844 5 -TAGGTYCACAMTGYGGTTCTTC-3 LPW1872 5 -AMCCTTGYTTAGGTGCAATACCT-3 LPW1848 5 -TTAAAACTGTYTTAGTAAGTCC-3 LPW1874 5 -TAGTAAAACTAGTTAYAACACC-3 N LPW1887 5 -TAGTGGTATGGATACTGCCTTGT-3 LPW1890 5 -GCTTTAACATTTCAGMATTACCA-3 LPW1891 5 -CAGTGTTTTGGTAAAAGAGGACC-3 LPW1892 5 -TACCACCTAGTGTCGAATTAGG-3 Recently, we have described the discovery of a novel group 2 coronavirus, coronavirus HKU1 (CoV-HKU1), from a patient with pneumonia [19] . Complete genome sequencing and phylogenetic analysis revealed that CoV-HKU1 is a distinct group 2 coronavirus, with G + C content of 32%, the lowest among all known coronaviruses. Although preliminary screening also identifie an additional patient with pneumonia with CoV-HKU1 in her nasopharyngeal aspirate (NPA), the epidemiological and clinical features of CoV-HKU1-associated pneumonia remain to be determined. In the present study, using prospectively collected NPAs, we examined the prevalence of CoV-HKU1 in patients with community-acquired pneumonia during a 12-month period. The clinical, laboratory, and radiological characteristics of patients with CoV-HKU1-associated pneumonia were described. The molecular epidemiological profil of CoV-HKU1 was also analyzed.
All prospectively collected NPAs from patients with community-acquired pneumonia that were sent to the clinical microbiology laboratories of 4 hospitals in Hong Kong during a 12-month period (22 March 2003 [the beginning of the SARS epidemic in Hong Kong] to 21 March 2004) for detection of SARS-CoV and were found to be negative for SARS-CoV RNA, by reverse-transcription polymerase chain reaction (RT-PCR) [20] , were included in the study. Community-acquired pneumonia is de- [16] .
To determine possible risk factors associated with CoV-HKU1-associated pneumonia, 2 age-and sex-matched controls per patient with CoV-HKU1-associated pneumonia were randomly selected from patients with community-acquired pneumonia whose NPAs were found to be negative for CoV-HKU1. Controls were within 5 years in age (older or younger) of the corresponding patients with CoV-HKU1-associated pneumonia and were admitted within 15 days before or after admission of the corresponding patients with CoV-HKU1-associated pneumonia. The hospital records, laboratory results, and chest ra- diographs of the controls were examined by 2 infectious disease physicians.
RNA extraction. Viral RNA was extracted from NPAs using the QIAamp Viral RNA Mini Kit (QIAgen) within 10 h of receipt of specimens. The eluted RNA (template for RT-PCR) was stored immediately at Ϫ70ЊC until use.
A 453-bp fragment of the pol gene of CoV-HKU1 was amplifie by RT-PCR using CoV-HKU1-specifi primers (LPW1926 [5 -AAAGGATGTTG-ACAACCCTGTT-3 ] and LPW1927 [5 -ATCATCATACTAAA-ATGCTTACA-3 ]) designed by multiple alignment of the nucleotide sequences of the pol genes of CoV-HKU1 [19] and those of other coronaviruses. RT was performed using the Su-perScript II kit (Invitrogen). The PCR mixture (50 mL) contained cDNA, PCR buffer, 200 mmol/L each dNTP, and 1.0 U of Taq polymerase (Boehringer). The mixtures were amplifie in 40 cycles of 94ЊC for 1 min, 48ЊC for 1 min, and 72ЊC for 1 min and a fina extension at 72ЊC for 10 min. To ensure the high specificit of the CoV-HKU1-specifi primers, RNA extracted from 200 NPAs positive for influenz A and B viruses, parainfluenz viruses 1-3, RSV, or adenovirus antigens and RNA from 12 NPAs positive for HCoV-229E, HCoV-OC43, HCoV-NL63, or SARS-CoV RNA (3 samples/coronavirus) were also subjected to RT-PCR using the 2 CoV-HKU1-specifi primers. The amplifie products were detected by agarose gel electrophoresis, as described elsewhere [19] .
Both strands of the PCR products were sequenced twice by use of an ABI Prism 3700 DNA Analyzer (Applied Biosystems), using the 2 PCR primers. The sequences of the PCR products were compared with the sequences of the pol genes of CoV-HKU1 [19] and those of other coronaviruses in the GenBank database.
ELISA using recombinant nucleocapsid (N) protein of CoV-HKU1. The ELISA-based IgG and IgM antibody tests were performed in accordance with our published protocol [19] . Each sample was tested in duplicate, and the mean absorbance for each serum sample was calculated.
The complete pol, S, and N genes of CoV-HKU1 from NPAs from 9 of the 10 patients from whom adequate amounts of RNA were available were amplifie and sequenced using the strategy described in our previous study [19] and the primers listed in table 1. The nucleotide and deduced amino acid sequences of the pol, S, and N genes were compared with those of CoV-HKU1 [19] and other group 2 coronaviruses. Phylogenetic tree construction was performed using the PileUp method with GrowTree (Genetics Computer Group).
Statistical analysis. A comparison of characteristics was made between patients with CoV-HKU1-associated pneumonia and those with non-CoV-HKU1-associated pneumonia and between patients who died of and those who survived CoV-HKU1-associated pneumonia. Fisher's exact test was used for categorical variables, and the Mann-Whitney U test was used for continuous variables.
was regarded as statistically P ! .05 significant
Clinical and laboratory characteristics. During the 12-month period, NPAs from 418 patients (men:women, 198:220; mean ‫ע‬ SD age, years) in 4 hospitals with community-ac-49 ‫ע‬ 26 quired pneumonia were found to be negative for SARS-CoV RNA by RT-PCR. A 453-bp fragment of the pol gene of CoV-HKU1 was amplifie and sequenced for 10 patients (2.4%). Sequence analysis revealed 0%-2% nucleotide differences between the sequences of the fragments and the sequence of the pol gene from The epidemiological, clinical, and radiological characteristics of the 10 patients with CoV-HKU1-associated community-acquired pneumonia are summarized in table 2. No epidemiological linkage was identifie among the 10 cases. All cases occurred during either spring or winter (January-May). The median age of these patients was 71.5 years (range, 13-96 years).
Seven were men, and 3 were women. Eight had underlying diseases, of whom 4 had underlying diseases of the respiratory tract. Four had recently traveled to southern China. Five were smokers. Clinically, the illness was not distinguishable from other community-acquired pneumonia illnesses. Fever, productive cough, and dyspnea were common symptoms at presentation. Upper respiratory tract symptoms were present in only 2 patients (patients 1 and 5). One patient (patient 7) had loose-stool diarrhea. Oxygen saturation levels on room air on admission were !95% in 2 patients. Airspace shadows were observed in the right lungs of 6 patients and in the left lungs of 6 patients. The upper, middle, and lower zones were affected in 2, 4, and 9 patients, respectively. All patients, except patient (table 3) . None of the 10 patients in this control group from whom serum samples were available had a 4-fold increase in IgG titer or the presence of IgM against CoV-HKU1. Two of the 10 patients died of CoV-HKU1-associated pneumonia. The firs patient (patient 2) was a 66-year-old man who had presented with dyspnea for 1 day. He had type 2 diabetes mellitus, old myocardial infarction, and gastric lymphoma with total gastrectomy in 2002 and was treated with chemotherapy. He had severe lymphopenia ( lymphocytes/L) and an 9 0.2 ϫ 10 oxygen saturation level of only 83% on admission. Chest radiographic examination revealed patchy airspace shadows in both lungs, with predominant involvement of the lower zones (figu e 2A). He died 11 days after admission. The other patient (patient 10) was a 72-year-old man who had presented with fever and productive cough for 1 week. He had type 2 diabetes mellitus, cerebrovascular accident, and prostatic carcinoma with bone metastasis complicated by spinal cord compression; laminectomy and Luque instrumentation were performed. He had lymphopenia ( lymphocytes/L), thrombocytopenia 9 0.9 ϫ 10 ( thrombocytes/L), deranged liver and renal function 9 33 ϫ 10 test results, and an oxygen saturation level of only 88% on admission. Chest radiographic examination revealed extensive airspace shadows in both lungs, with the middle zones more severely involved (figu e 2B). He died 5 days after admission.
The clinical, laboratory, and radiological characteristics of patients who survived and those who died of CoV-HKU1-associated community-acquired pneumonia were compared (table 4). Patients who died had lower hemoglobin concentrations (P p .04), monocyte counts (P p .04), serum albumin levels (P p .04), and oxygen saturation levels on admission (P p .03) and had bilateral involvement ( ) and more zones involved P p .003 ( ) on chest radiographs. P p .01
The complete pol, S, and N genes of CoV-HKU1 from NPAs from 9 of the 10 patients from whom adequate amounts of RNA were available were amplifie and sequenced. The phylogenetic trees and nonsynonymous mutations and the corresponding amino acid changes are shown in figu e 3. For the S gene, there were 317 and 306 nt positions with synonymous and nonsynonymous mutations, respectively (figu e 3B); 72% and 28% of the nonsynonymous mutations were in the putative S1 and S2, respectively (figu e 4). For the N gene, there were 42 and 53 nt positions with synonymous and nonsynonymous mutations, respectively (figu e 3C). The nucleotide sequences of 7 of the 9 S or N genes showed similar sequences (genotype A), and those of the other 2 genes also showed similar sequences (genotype B) (figu e 3B and 3C). For the CoV-HKU1 specimen from patient 1, 2 peaks (T and C) were consistently observed at nt 1300 of the N gene, suggesting the presence of a quasi species (figu e 3C). For the pol gene, there were 95 and 13 nt positions with synonymous and nonsynonymous mutations, respectively (figu e 3A). The nucleotide sequences of the pol genes in the 7 CoV-HKU1 specimens of genotype A were also clustered together (figu e 3A). Interestingly, the 7 CoV-HKU1 specimens of genotype A were from 7 patients with underlying diseases, and the 2 specimens of genotype B were from the 2 patients without underlying diseases (table 2) . Furthermore, multiple alignments of the nucleotide sequences of the pol genes from the 9 CoV-HKU1 specimens and those from the HCoV-OC43, HCoV-229E, HCoV-NL63, and SARS-CoV supported the notion that the primers used in the present study were specifi for CoV-HKU1.
CoV-HKU1, a novel group 2 coronavirus, is associated with community-acquired pneumonia. Since the SARS epidemic in 2003, we have started to prospectively collect NPAs from patients with community-acquired pneumonia and store the extracted RNA, so that, when a novel virus is discovered, the epidemiological and, hence, the clinical, laboratory, and radiological features of the disease can be studied in a timely manner. In January 2004, we discovered a novel coronavirus, CoV-HKU1, from a patient with community-acquired pneumonia [19] . The RNA extracted from prospectively collected NPAs was immediately retrieved, and the presence of CoV-HKU1 was investigated. Ten of the 418 NPAs were positive for CoV-HKU1, giving an incidence of 2.4%. The presence of CoV-HKU1 in these specimens was genuine-that is, it was not due to contamination-since amplificatio and sequencing of multiple genes (pol, S, and N) of CoV-HKU1 indicated the presence of CoV-HKU1 with different nucleotide sequences from the different patients. Moreover, the clinical significanc of CoV-HKU1 was confi med by the presence of a specifi antibody response in all 6 patients from whom serum samples were available. The antibody response in these patients was unlikely to be the result of cross-reaction with other known human coronaviruses, since none of the 20 serum samples obtained from patients with HCoV-OC43 infections or SARS-CoV pneumonia had positive results in the recombinant N ELISA (authors' unpublished data). Further studies on detection of CoV-HKU1 in NPAs from healthy individuals will determine whether asymptomatic shedding of CoV-HKU1 occurs.
Similar to HCoV-229E, HCoV-OC43, and HCoV-NL63, CoV-HKU1 is a human coronavirus that is endemic in humans. Similar to other human coronavirus infections, cases of CoV-HKU1-associated pneumonia occurred during winter and spring. Most patients with CoV-HKU1-associated pneumonia were old (80% were 165 years old) and had major underlying diseases, especially those of the respiratory and cardiovascular systems. To study the phylogeny and relationships among the 10 CoV-HKU1 specimens, we sequenced the pol, S, and N genes from the 9 CoV-HKU1 specimens that contained adequate amounts of RNA. Combined with the data on partial sequencing of the pol genes from the 10 CoV-HKU1 specimens (figu e 1), results showed that, unlike the epidemiological profil of SARS-CoV, the 10 CoV-HKU1 specimens were not clonal, and the topology of the phylogenetic trees did not follow the pattern of a clonal outbreak (figu e 3). Interestingly, the phylogenetic trees constructed using the sequences of both the S and N genes showed that CoV-HKU1 of genotype B was detected in NPAs from with 2 patients without underlying diseases, but CoV-HKU1 of genotype A was detected in NPAs from patients with underlying diseases (figu e 3B and 3C). Sequencing of more CoV-HKU1 specimens may reveal the presence of genotypes or clades of CoV-HKU1 with differential virulence.
Compared with SARS-CoV pneumonia, CoV-HKU1-associated pneumonia is a monophasic disease, and most patients had relatively mild symptoms that were localized to the respiratory tract and were, therefore, hospitalized only briefl . SARS-CoV pneumonia is often described as a biphasic disease, with the firs phase due to cell lysis as a result of viral replication and the second phase due to immunopathological damage [4, 12] . On the other hand, all 10 patients with CoV-HKU1-associated pneumonia showed the pattern of a monophasic disease. Although dyspnea was present in one-half of the patients with CoV-HKU1-associated pneumonia at presentation, compared with only ∼20% of patients with SARS-CoV pneumonia at presentation [4] , patients with CoV-HKU1-associated pneumonia often recovered quickly, but patients with SARS-CoV pneumonia deteriorated after 7-10 days [4, 12] . For the 8 patients who recovered, the median duration of hospitalization was only 5.5 days. This rapid recovery of patients with CoV-HKU1-associated pneumonia could be related to the rapid control of the virus by the immune system. This is in line with the results of our previous study, which showed that the index patient with CoV-HKU1-associated pneumonia had his peak viral load at around day 3 after the onset of illness [19] . Moreover, only 1 of the patients had extrapulmonary symptoms, and all available extrapulmonary specimens (stool, urine, and serum) were negative, by RT-PCR, for CoV-HKU1 (authors' unpublished data). On the other hand, for SARS-CoV pneumonia, patients usually had their peak viral loads 7-10 days after onset of illness [12] . Furthermore, the virus can be readily detected in extrapulmonary specimens, in which the viral loads correlated with the manifestations in the corresponding systems [22] . These finding imply that the virus was not well controlled by the immune system during the initial phase of the illness.
Despite the relatively mild course of disease in most patients, CoV-HKU1-associated pneumonia is associated with mortality in a minority of patients who have lower hemoglobin concentrations, monocyte counts, serum albumin levels, and oxygen saturation levels on admission and more-extensive involvement on chest radiographs. As in most cases of pneumonia, extensive involvement in the lungs will result in poor gaseous exchange and, hence, hypoxia and eventually death. The lower hemoglobin concentrations, monocyte counts, and serum albumin levels could represent poorer premorbid states and narrower margins to figh against infections. Both patients who died had underlying diabetes mellitus, malignancy (gastric lymphoma in one and prostate carcinoma in the other), and cardiovascular disease (old myocardial infarction in one and cerebrovascular accident in the other).