Evidence supporting a zoonotic origin of human coronavirus strain NL63 - PubMed (original) (raw)
. 2012 Dec;86(23):12816-25.
doi: 10.1128/JVI.00906-12. Epub 2012 Sep 19.
Shimena Li, Boyd Yount, Alexander Smith, Leslie Sturges, John C Olsen, Juliet Nagel, Joshua B Johnson, Sudhakar Agnihothram, J Edward Gates, Matthew B Frieman, Ralph S Baric, Eric F Donaldson
Affiliations
- PMID: 22993147
- PMCID: PMC3497669
- DOI: 10.1128/JVI.00906-12
Evidence supporting a zoonotic origin of human coronavirus strain NL63
Jeremy Huynh et al. J Virol. 2012 Dec.
Abstract
The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans.
Figures
Fig 1
Phylogeny of the mitochondrial cytochrome b gene from several mammalian species. This maximum likelihood tree shows that North American bats of the family Vespertilionidae are more closely related to themselves than to other bat or common mammalian species. EpFu, big brown bat; PeSu, tricolored bat; MyLu, little brown myotis; MyLe, eastern small-footed myotis; MySe, northern long-eared myotis. The scale bar represents nucleotide substitutions. Only nodes with bootstrap support above 70% are labeled.
Fig 2
A novel α-CoV in the tricolored bat is closely related to HCoV-NL63. Novel α-CoV sequences have been found in the fecal samples of several North American bat species. (A) Schematic showing where the two fragments (used for the trees in panels B and C) occur in the NL63 genome. (B) A >2.2-kb fragment of the replicase region (starting at position 16480 in the schematic) was sequenced from three samples, including two from the big brown bat (NECoV and ARCoV.1) and one from the tricolored bat (ARCoV.2). A maximum likelihood tree comparing the nucleotide sequences of these bat CoVs to other known CoVs indicated that NECoV and ARCoV.1 are very closely related while ARCoV.2 is significantly different from NECoV and ARCoV.1. The three novel α-CoV sequences form a novel cluster in the α-CoV group. (C) Molecular clock analysis using a 650- to 800-nt portion of the highly conserved replicase region (starting at position 13810 in panel A) predicted that the MRCA of bat CoVs from the Hipposideros caffer ruber bats and HCoV-229E was likely to have existed 212 to 350 years ago (in agreement with Pfefferle et al., 2009 [36]). The MRCA for HCoV-NL63 and ARCoV.2 was predicted to have existed 563 to 822 years ago. NECOV and ARCoV.1 were identical in this region, so only ARCoV.1 is shown, and it clustered with other bat α-CoVs.
Fig 3
Immortalization of tricolored bat lung cells, PESU-B5L. Primary tricolored bat lung cells were grown in enriched growth medium and passaged four times to generate a suitable quantity for immortalization. Primary cells were immortalized with lentiviral vectors containing hTERT and the Bmi-1 proto-oncogene. (A) Primary tricolored bat lung cells. (B) Primary PESU-B5L lung cells reached senescence by passage 10. (C) Immortalized cells at passage 10, including 4 passages as primary cells and 6 passages postimmortalization. The immortalized PESU-B5L cells were still going strong after more than 30 passages (over 100 doublings).
Fig 4
Infection of PESU-B5L cells with human coronavirus NL63 expressing GFP. PESU-B5L cells were infected with mock or NL63gfp at an MOI of 0.25 and monitored by bright-field and fluorescence microscopy. (A and B) Mock-infected cells by bright-field (A) and fluorescence (B) microscopy. (C through F) Cells infected with NL63gfp visualized at day 3 postinfection by bright-field microscopy (C) and by fluorescence microscopy (D) and at day 5 postinfection by bright-field microscopy (E) and by fluorescence microscopy (F).
Fig 5
Verification of NL63 replication in PESU-B5L cells. Total RNA was harvested from cells on day 5 postinfection and subjected to RT-PCR using primers designed to detect leader-containing transcripts of the N gene, which would be present only if NL63 replicated. (A) Subgenomic transcripts were detected in the PESU-B5L cells infected with both wtNL63 and NL63gfp. (B) The sgN band for wtNL63 was excised, and the DNA was purified and sequenced to verify that the HCoV-NL63 leader sequence and the 5′ end of the N gene were present. L, ladder; N, wt-NL63; G, NL63gfp; M, mock. The leader sequence has a beige bar beneath it, and the 5′ end of the N gene has a green bar beneath it.
Fig 6
Evidence of HCoV-NL63 infection in PESU-B5L cells. PESU-B5L cells were infected with HCoV-NL63 at an MOI of 0.8. At 72 h postinfection, cells were probed for the nucleocapsid protein of HCoV-NL63 by immunofluorescence assay and Western blotting. (A) I, mock-infected cells, fluorescence microscopy; II, mock-infected cells, bright-field microscopy; III, HCoV-NL63-infected cells, fluorescence microscopy; IV, HCoV-NL63-infected cells, bright-field microscopy. (B) Western blot of HCoV-NL63 and mock-infected PESU-B5L cell lysates with β-actin as a loading control. Nucleocapsid (N) protein appears as a distinct band at ∼45 kDa.
Fig 7
Evidence of SARS-CoV replication in PESU-B5L cells. PESU-B5L cells were infected with SARS-CoV at an MOI of ∼1, and total RNA was harvested from the cells at 24-h intervals and subjected to RT-PCR using primers designed to detect leader-containing transcripts of the subgenomic GFP (sgGFP), indicative of SARS-CoV replication. Subgenomic transcripts were detected at 24 h postinfection but disappeared by 48 h postinfection, suggesting that SARS-CoV replicates in these cells at a low level. L, Promega 1-kb ladder; M, mock-infected cells; V, SARS-CoV-infected cells.
Fig 8
Evidence of SARS-CoV replication in PESU-B5L cells. PESU-B5L cells were infected with the mouse-adapted strain of SARS-CoV, MA15, and with chimeric SARS-CoV viruses bearing the spike genes of GD03 and HC/SZ/61/03 at an MOI of ∼1. Total RNA was harvested from the cells at 48 and 72 h postinfection and subjected to RT-PCR using primers designed to detect leader-containing subgenomic transcripts indicative of replication. (A) Subgenomic (sg) transcripts for X1, envelope (E), membrane (M), and open reading frame 6 [ORF6 (X3)] were detected at both time points for all three strains. Lane 1, MA15 at 48 h; lane 2, MA15 at 72 h; lane 3, Invitrogen 1-kb Plus ladder; lane 4, SARS+GD03 at 48 h; lane 5, SARS+GD03 at 72 h; lane 6, mock-infected cells; lane 7, SARS+HC/SZ/61/03 at 48 h; and lane 8, SARS+HC/SZ/61/03 at 72 h. (B) The sg M bands for all three strains were excised, and the DNA was purified and sequenced to verify that the SARS-CoV leader sequence and the 5′ end of the M gene were present. Shown here is sg M from SARS-CoV strain GD03.
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