Culturing the Unculturable: Human Coronavirus HKU1 Infects,Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is critical to develop suitable in vitro platforms to isolate and characterize the replication kinetics and pathogenesis of recently identified human pathogens. HCoV-HKU1, a human coronavirus identified in a clinical sample from a patient with severe pneumonia, has been a major challenge for successful propagation on all immortalized cells tested to date. To determine if HCoV-HKU1 could replicate in in vitro models of human ciliated airway epithelial cell cultures (HAE) that recapitulate the morphology, biochemistry, and physiology of the human airway epithelium, the apical surfaces of HAE were inoculated with a clinical sample of HCoV-HKU1 (Cean1 strain). High virus yields were found for several days postinoculation and electron micrograph, Northern blot, and immunofluorescence data confirmed that HCoV-HKU1 replicated efficiently within ciliated cells, demonstrating that this cell type is infected by all human coronaviruses identified to date. Antiserum directed against human leukocyte antigen C (HLA-C) failed to attenuate HCoV-HKU1 infection and replication in HAE, suggesting that HLA-C is not required for HCoV-HKU1 infection of the human ciliated airway epithelium. We propose that the HAE model provides a ready platform for molecular studies and characterization of HCoV-HKU1 and in general serves as a robust technology for the recovery, amplification, adaptation, and characterization of novel coronaviruses and other respiratory viruses from clinical material.About 335 new or emerging infectious diseases have been identified since 1940 (23), and while many threaten human health, the global economy, and national security, respiratory pathogens are of particular public health concern. Using modern methods, several previously unknown viruses have been identified, including respiratory pathogens (1, 18, 27, 54, 57), yet research remains restricted to prevalence and disease association studies since a virus culture system is oftentimes lacking. Immortalized tissue culture cells are adapted to growth in laboratory conditions and, as such, display altered gene expression patterns, which may not be optimal for the replication of fastidious viruses. Primary cell-differentiated culture models provide alternative in vitro model systems closer in nature to the in vivo host tissue environment for infection studies and amplification of pathogens for further characterization. Here, we use an in vitro model of human ciliated airway epithelial cell cultures (HAE) that mimic the properties of the cartilaginous airway epithelium (17) to culture the previously unculturable human coronavirus HKU1 (HCoV-HKU1).Coronaviruses are important pathogens of humans and animals, causing a range of symptoms depending on the host. Following the severe acute respiratory syndrome (SARS)-CoV epidemic, several new strains of human coronaviruses were identified by molecular techniques, including HCoV-NL63, identified in the Netherlands from an infant with bronchiolitis (54), and HCoV-HKU1, identified in an adult patient with severe pneumonia in Hong Kong (57). HCoV-NL63 has been demonstrated to infect and replicate in both conventional immortalized cells and human ciliated airway cell cultures, producing sufficient amounts of virus for characterization studies of viral replication and pathogenesis and the successful development of an infectious clone (3, 13, 22, 41). In contrast, little is known about HCoV-HKU1, as no in vitro replication model has been identified to date, limiting further investigations of the virus.Clinical isolates of previously isolated human coronaviruses have been adapted to replicate in standard transformed cell culture; for example, SARS-CoV and HCoV-NL63 replicate efficiently in epithelial monkey kidney cells (VeroE6 and LLC-MK2), HCoV-OC43 in BHK21 cells, and HCoV-229E in MRC5 cells (14, 24, 35, 47, 54, 59). Despite the successful amplification of these human coronaviruses in cell lines, all attempts to date to culture a clinical isolate of HCoV-HKU1 have failed. No HCoV-HKU1 genomic replication was observed after inoculation of standard cell lines previously utilized for virus propagation, including RD (human rhabdomyosarcoma cells), HRT-18 (colorectal adenocarcinoma cells), HEp-2 (human epithelial carcinoma cells), MRC-5 (human lung fibroblast cells), A549 (human lung epithelial adenocarcinoma cells), Caco2 (human colorectal adenocarcinoma cells), Huh-7 (human hepatoma cells), B95a (marmoset B-lymphoblastoid cells), mixed neuron-glia culture, LLC-MK2 (rhesus monkey kidney cells), FRhK-4 (rhesus monkey kidney cells), BSC-1 (African green monkey kidney cells), Vero E6 (African green monkey kidney cells), MDCK (Madin-Darby canine kidney cells), I13.35 (murine macrophage cells), and L929 (murine fibroblast cells) (57).Here, we use human ciliated airway epithelial cell cultures to successfully propagate HCoV-HKU1 for the first time in vitro. In this culture model, HCoV-HKU1 genome copy numbers increased by several logs over the initial three-day incubation period and electron micrograph, Northern blot, and immunofluorescence data confirmed HKU1 replication in HAE and that ciliated cells were the preferential target for virus infection, the same cell type infected by all human coronaviruses tested so far in these model systems.