Culturing the unculturable: human coronavirus HKU1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures - PubMed (original) (raw)

Culturing the unculturable: human coronavirus HKU1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures

Krzysztof Pyrc et al. J Virol. 2010 Nov.

Abstract

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.

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Figures

FIG. 1.

FIG. 1.

HCoV-HKU1 genome and subgenomic RNA species schematic. All open reading frames (ORFs) in the HCoV-HKU1 genome are shown as rectangles in the top schematic, and the proteins expressed from each are indicated. Viral leader sequences are shown as small yellow rectangles at the 5′ end of each line representation of the viral subgenomic RNA species at the bottom of the figure. The 5′-most ORF is translated from each subgenomic RNA, and this ORF is indicated as a colored rectangle for each RNA. UTR, untranslated region; HE, hemagglutinin; S, spike; AP, accessory open reading frame/protein; E, envelope; M, membrane; N, nucleocapsid.

FIG. 2.

FIG. 2.

(A) Replication of an HCoV-HKU1 clinical isolate in HAE. HAE were inoculated with diluted nasal aspirates for the first passage (108 copies/ml) and with apical washes from 96 h postinoculation for the subsequent passage. The bars represent real-time PCR analysis of apical media harvested from HCoV-HKU1-infected HAE at 96 h postinoculation or from the inoculating clinical sample. (B) Replication kinetics of HCoV-HKU1 in HAE. Cultures were inoculated with passage 3 virus (108 copies/ml). Data points represent real-time PCR of apical washes from HCoV-HKU1-infected HAE, harvested at the indicated times postinoculation. Data are presented as HCoV-HKU1 RNA copies/ml and are representative of results from experiments performed in duplicate.

FIG. 3.

FIG. 3.

HCoV-HKU1 RNA species present during infection of HAE. Representative Northern blot analysis for total RNA isolated from HCoV-HKU1-infected HAE. RNA species are indicated by arrows to the right of the autoradiograph. HE, hemagglutinin; S, spike; ORF 4, accessory open reading frame; E, envelope; M, membrane; N, nucleocapsid; N2, internal ORF in N. (A) Total RNA, 0.1 μg; (B) total RNA, 1 μg.

FIG. 4.

FIG. 4.

HCoV-HKU1 subgenomic species. (A) Subgenomic mRNA species generated during HCoV-HKU1 replication in HAE. Each lane contains RT-PCR products from total RNA isolated from HCoV-HKU1-inoculated HAE, amplified using a 5′ HCoV-HKU1 leader primer and the indicated ORF-specific 3′ primer. Size markers are indicated on the far left of the gel, and the size of each band is shown at the base of each well. 1a, open reading frame 1a replicase proteins; HE, hemagglutinin; S, spike; AP, accessory open reading frame 4/protein; E, envelope; M, membrane; N, nucleocapsid. (B) Kinetics of subgenomic RNA species synthesis during HCoV-HKU1 replication in HAE. Each lane contains RT-PCR bands from RNA extracted from HCoV-HKU1-inoculated HAE for the first 500 nt of the structural proteins spike, membrane, and nucleocapsid. Marker sizes are indicated between the gels, times postinoculation are indicated above each well, and the gene of interest is indicated at the base of each gel. S, spike; M, membrane; N, nucleocapsid. (C) Leader-body junctions of all HCoV-HKU1 sg mRNAs. Shown on the top row is the leader (L) sequence, and the bottom row shows the specific sequences upstream of the structural genes (G). The sequence in the middle (sg) represents the mature sg mRNA generated during coronavirus replication. Sequence homology between the strands near the junction is highlighted in black. HE, hemagglutinin; S, spike; ORF4, accessory open reading frame; E, envelope; M, membrane; N, nucleocapsid; N2, internal ORF in N.

FIG. 5.

FIG. 5.

Maximum likelihood cladogram of HKU1 genomes. HCoV-HKU1 strain Caen1 was compared to 23 additional full-length HCoV-HKU1 virus genomes to determine its evolutionary relatedness within the HCoV-HKU1 lineage. This maximum likelihood tree generated with the PhyML package shows that HCoV-HKU1 strain Caen1 is most closely related to the A genotype. Branch points are labeled with bootstrap values, based upon 100 iterations, and the cladogram is set to be proportional.

FIG. 6.

FIG. 6.

HCoV-HKU1 infects ciliated cells of HAE. Representative images of HCoV-HKU1 immunoreactivity in histological sections of HAE 72 h postinoculation with HCoV-HKU1. Histological sections were probed with antisera directed against mouse hepatitis virus (MHV) whole virions (green) and β-tubulin IV (red). (A) HCoV-HKU1 immunoreactivity (green) in histological sections, demonstrating that HCoV-HKU1 infects ciliated cells. (B) HCoV-HKU1-inoculated HAE probed with preimmune rabbit serum and β-tubulin IV antibody, demonstrating no HCoV-HKU1 immunoreactivity. Original magnification, ×40. Scale bars, 100 μm; red arrows, cilial shafts of ciliated cells; green arrow, HKU1-infected cells; white arrows, basal cells present on the Transwell supports. Colocalization of HCoV-HKU1 and ciliated cell immunoreactivity (greenish-yellow) indicate ciliated cell infection by HCoV-HKU1.

FIG. 7.

FIG. 7.

Ultrastructural localization of HCoV-HKU1 in HAE. Representative transmission electron photomicrographs of HAE inoculated with HCoV-HKU1. (A and B) HAE mock inoculated with the vehicle alone, demonstrating the typical morphological features of the apical surfaces of HAE with cilia (black arrows) and microvilli (white arrows). (C to G) HAE infected with HCoV-HKU1 for 96 h showing the presence of the large numbers of virions (circled) associated with the surfaces of ciliated cells or shed into pericilial regions (black arrows, cilia; white arrows, microvilli). Intracellular virions were also noted inside vesicular structures in the cytoplasm of ciliated cells (D, box). F represents a high-power image of a virion associated with the tip of a microvillus. Scale bars are shown in the lower right of each panel and represent 2 μm in panels A to D and G and 1 μm in panels E and F.

FIG. 8.

FIG. 8.

Efficient infection of HAE by HCoV-HKU1 does not require HLA-C. Replication kinetics of HCoV-HKU1 were assessed by real-time RT-PCR of RNA present in apical washes from HCoV-HKU1-infected HAE cultures pretreated with either no antibody (yellow), HLA-C-specific antibody (blue), HLA-BC antibody (pink), a combination of the antibodies (green), or ACE2 antibody (black). Titers are represented by virus copies per milliliter. No significant differences in HCoV-HKU1 infection and replication were measured in any treatment group. n = 3 for each treatment group.

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