A novel coronavirus, HKU1, from patients with pneumonia

Abstract number: 1133_46

Woo P.C.Y., Lau S.K.P., Chu C.-M., Chan K.-H., Tsoi H.-W., Huang Y., Wong B.H.L., Poon R.W.S., Cai J.J., Luk W.-K., Poon L.L.M., Guan Y., Wong S.S.Y., Peiris J.S.M., Yuen K.-Y.

Objectives:  

To characterize and analyze the complete genome of a novel coronavirus, coronavirus HKU1 (CoV-HKU1), from patients with pneumonia.

Methods:  

We amplified a 440-bp fragment of the RNA-dependent RNA polymerase (pol)gene of coronaviruses by RT-PCR, using coronavirus conserved primers, from RNA extracted from the nasopharyngeal aspirate (NPA) of a 71-year old Chinese man with pneumonia. The complete genome of the coronavirus (CoV-HKU1) was amplified and sequenced using RNA extracted from the NPA as template and degenerate primers designed by multiple alignment of the genomes of other group 2 coronaviruses. Real-time quantitative RT-PCR was performed to quantify the amount of virus in the NPA obtained in the first to the fifth week of the illness. Specific antibody was detected by Western blot assay and ELISA using recombinant nucleocapsid of CoV-HKU1. Screening for CoVHKU1 on NPA obtained during the SARS period was carried out by RT-PCR.

Results:  

RT-PCR and sequencing of the 440-bp fragment of the pol gene showed that there were 89–91% amino acid identity between the sequence of the fragment and those of other group 2 coronaviruses. The genome of CoV-HKU1 is a 29926-nucleotide, polyadenylated RNA. The G + C content is 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoVHKU1 is a new group 2 coronavirus. Quantitative RT-PCR showed that the amount of CoV-HKU1 RNA were 8.5 to 9.6 × 10⁶ copies per ml in his NPA 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 IgM titres of 1:20, 1:40 and 1:80 and IgG titres of <1:1000, 1:2000 and 1:8000 in the first, second and fourth weeks of the illness respectively. Cultural, antigenic, RNA, or serological detection of other microbial pathogens were negative. Screening of 400 NPA collected during the SARS period identified the presence of CoV-HKU1 RNA in one specimen, with a viral load of 1.13 × 10⁶ copies per ml, from a 35-year old woman with pneumonia.

Conclusion:  

Our data support the existence of a novel group 2 coronavirus in the NPA of patients with pneumonia. Resolution of the illness in the index patient was associated with the disappearance of the viral RNA and the development of specific IgG and IgM antibody response against the virus.