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. 2019 Nov 26;93(24):e01448-19.
doi: 10.1128/JVI.01448-19. Print 2019 Dec 15.

Broad Cross-Species Infection of Cultured Cells by Bat HKU2-Related Swine Acute Diarrhea Syndrome Coronavirus and Identification of Its Replication in Murine Dendritic Cells In Vivo Highlight Its Potential for Diverse Interspecies Transmission

Yong-Le Yang #  1 , Pan Qin #  1 , Bin Wang  1 , Yan Liu  1 , Guo-Han Xu  1 , Lei Peng  1 , Jiyong Zhou  1 , Shu Jeffrey Zhu  2 , Yao-Wei Huang  2
Affiliations

Broad Cross-Species Infection of Cultured Cells by Bat HKU2-Related Swine Acute Diarrhea Syndrome Coronavirus and Identification of Its Replication in Murine Dendritic Cells In Vivo Highlight Its Potential for Diverse Interspecies Transmission

Yong-Le Yang et al. J Virol. .

Abstract

Outbreaks of severe diarrhea in neonatal piglets in Guangdong, China, in 2017 resulted in the isolation and discovery of a novel swine enteric alphacoronavirus (SeACoV) derived from the species Rhinolophus bat coronavirus HKU2 (Y. Pan, X. Tian, P. Qin, B. Wang, et al., Vet Microbiol 211:15-21, 2017). SeACoV was later referred to as swine acute diarrhea syndrome CoV (SADS-CoV) by another group (P. Zhou, H. Fan, T. Lan, X.-L. Yang, et al., Nature 556:255-258, 2018). The present study was set up to investigate the potential species barriers of SADS-CoV in vitro and in vivo We first demonstrated that SADS-CoV possesses a broad species tropism and is able to infect cell lines from diverse species, including bats, mice, rats, gerbils, hamsters, pigs, chickens, nonhuman primates, and humans. Trypsin contributes to but is not essential for SADS-CoV propagation in vitro Furthermore, C57BL/6J mice were inoculated with the virus via oral or intraperitoneal routes. Although the mice exhibited only subclinical infection, they supported viral replication and prolonged infection in the spleen. SADS-CoV nonstructural proteins and double-stranded RNA were detected in splenocytes of the marginal zone on the edge of lymphatic follicles, indicating active replication of SADS-CoV in the mouse model. We identified that splenic dendritic cells (DCs) are the major targets of virus infection by immunofluorescence and flow cytometry approaches. Finally, we demonstrated that SADS-CoV does not utilize known CoV receptors for cellular entry. The ability of SADS-CoV to replicate in various cells lines from a broad range of species and the unexpected tropism for murine DCs provide important insights into the biology of this bat-origin CoV, highlighting its possible ability to cross interspecies barriers.IMPORTANCE Infections with bat-origin coronaviruses (CoVs) (severe acute respiratory syndrome CoV [SARS-CoV] and Middle East respiratory syndrome CoV [MERS-CoV]) have caused severe illness in humans after "host jump" events. Recently, a novel bat-HKU2-like CoV named swine acute diarrhea syndrome CoV (SADS-CoV) has emerged in southern China, causing lethal diarrhea in newborn piglets. It is important to assess the species barriers of SADS-CoV infection since the animal hosts (other than pigs and bats) and zoonotic potential are still unknown. An in vitro susceptibility study revealed a broad species tropism of SADS-CoV, including various rodent and human cell lines. We established a mouse model of SADS-CoV infection, identifying its active replication in splenic dendritic cells, which suggests that SADS-CoV has the potential to infect rodents. These findings highlight the potential cross-species transmissibility of SADS-CoV, although further surveillance in other animal populations is needed to fully understand the ecology of this bat-HKU2-origin CoV.

Keywords: Coronavirus; SADS-CoV; interspecies transmission; mouse infection model.

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Figures

FIG 1
FIG 1
Immunofluorescence assay showing susceptibility of different cell lines to SADS-CoV infection. Immunofluorescence assay of cells infected with SADS-CoV at an MOI of 0.01 was performed using rabbit anti-SADS-CoV-M polyclonal Ab (200× magnification) and Alexa Fluor 488-conjugated anti-rabbit IgG as secondary antibody, with DAPI for visualization of cell nuclei. Mock-infected cells were treated with the same procedures as appropriate. Cells were tested from different species of origin, including bats (BFK and Tb-1) (A), hamsters (CHO and BHK-21) (B), mice (NIH/3T3 and RAW264.7) (C), rats (BRL-3A and NRK-52E) (D), humans (Huh-7, HepG2, 293T, A549, and HeLa) (E), monkeys (Marc-145, Cos-7, BSC-40, and Vero) (F), pigs (ST, PK15, LLC-PK1, and IPEC-J2) (G), chickens (DF-1) (H), dogs (MDCK) (I), and gerbil primary kidney cells (J).
FIG 2
FIG 2
Growth of SADS-CoV in different cell lines through 5 days postinfection. To determine the effect of trypsin on SADS-CoV infection, each cell line was infected in the following three conditions: “no trypsin” treatment, inoculated with SADS-CoV diluted in maintenance medium (MM) for 2 h and subsequently replaced with MM (A); “pretrypsin” treatment, inoculated with SADS-CoV diluted in MM containing 5 μg/ml trypsin (MMT) for 2 h and subsequently replaced with MM (B); and “double-trypsin” treatment, inoculated with SADS-CoV in MMT and subsequently replaced with MMT (C). Infection supernatants were collected at 12, 24, 36, 48, 72, and 120 hpi for viral load detection by a qRT-PCR assay targeting the viral N gene. Data are expressed as the mean viral load (log10 copies/μl) ± standard deviation (SD), and all experiments were performed in triplicate. The 293T, CHO, BRL-3A, and NRK-52E cell lines did not survive in the presence of trypsin. (D) Infectious titers (TCID50/ml) of SADS-CoV secreted from HeLa, Vero, Tb-1, BHK-21 PK-15, and MDCK cells were determined on Vero cells.
FIG 3
FIG 3
SADS-CoV infection of mice. C57BL/6J WT mice were infected per orally (p.o.; black) or intraperitoneally (i.p.; red) with 5 × 105 TCID50 of purified SADS-CoV. Viral loads in different tissue samples, including stomach (A), small intestinal segments (B), large intestinal segments (C), lymphoid tissues (D), the other organs (liver, kidney, heart, and lung) (E), and feces (F) collected at 1, 3, 5, 7, 14, 21, and 28 days postinfection (dpi) were determined by qRT-PCR. MLN, mesenteric lymph nodes. Data are from three independent experiments, and each symbol represents titers from an individual sample (*, P < 0.05). The limit of detection was 1 × 102 genome copies/mg. (G) SADS-CoV IgG antibodies were detected in serum samples collected at euthanasia using an ELISA based on purified SADS-CoV virus particles.
FIG 4
FIG 4
SADS-CoV replication in mouse splenocytes. Hematoxylin and eosin staining (HE) and immunohistochemistry (IHC) were performed on sections of spleen from intraperitoneally infected mice (A) and Mock-infected mice at 3 dpi (B) using anti-dsRNA antibodies to identify splenic cells that support active virus replication. (C) SADS-CoV infection could also be detected by IHC using SADS-CoV nonstructural protein antibody (anti-AC) and structural protein antibody (anti-M). Scale bars = 50 μm, except for magnified fields shown on the right side, with scale bars = 10 μm. (D) SADS-CoV antibody validation in Vero cells for developing the flow cytometry assay. Flow cytometry plots of Vero cells infected with SADS-CoV (MOI, 0.1) at 24 hpi; staining with anti-N or anti-AC. (E) Flow cytometry detection of Nsp3-AC antigens of SADS-CoV in splenocytes from infected mice using anti-AC antibody at 3 dpi. The data are presented as the fold increase in staining splenocytes from infected mice relative to the mock-infected group for statistical purposes (left); *, P < 0.05. (F) Representative FACS plots of (E). The solid-line frame-gated anti-AC positive splenocytes from p.o. or i.p. inoculated mice. The plot of mock-infected cells stained only with secondary antibody was also shown. (G) Isolated mouse splenocytes were infected with SADS-CoV at an MOI of 1, and SADS-CoV N protein expression was detected by IFA with anti-N antibody. (H) Flow cytometry detection of nonstructural protein antigens of SADS-CoV in infected splenocytes at 48 hpi using anti-AC antibody. The data are presented as the fold increase in staining cells relative to the mock-infected cells for statistical purposes (left); *, P < 0.05. (I) Growth of SADS-CoV in ex vivo splenocytes was monitored over 72 hpi by qRT-PCR targeting the SADS-CoV N gene.
FIG 5
FIG 5
SADS-CoV infection of dendritic cells in the spleen of mice inoculated via i.p. route. (A) Splenocytes were extracted from infected mice at 3 dpi, and flow cytometry was used to detect nonstructural antigen AC of SADS-CoV with immune cell markers on splenocytes, including B cells (CD19+), T cells (CD4+), macrophages (F4/80+), and dendritic cells (DCs; CD11c+). The data are presented as the fold increase in positive staining cells from infected mice relative to the mock-infected cells for statistical purposes; ***, P < 0.001. (B) Immunofluorescence assay of SADS-CoV dsRNA and DC marker CD11c in sections of spleen from intraperitoneally (i.p.) infected mice (B) and mock-infected mice (C) at 3 dpi. (D) SADS-CoV infection could also be detected by IFA using anti-AC and anti-M antibodies. The numbers of SADS-CoV-positive DCs in i.p.-infected mice were counted and averaged from 10 to 15 different visual fields (E), and the proportion of DCs in infected cells was presented with a Venn diagram (F). Scale bars = 50 μm, except for magnified fields shown on the right side, with scale bars = 10 μ m.
FIG 6
FIG 6
SADS-CoV utilizes an unknown receptor for cellular entry. (A) MDCK cells overexpressing each of the four known CoV receptors fused with detectable tags (pAPN-Flag, hDPP4-Flag, mCEACAM1a-Flag, or hACE2-GFP) did not confer SADS-CoV infection at 24 h posttransfection of the expression plasmids. At 48 h, SADS-CoV-inoculated cells transfected with pAPN-Flag, hDPP4-Flag, or mCEACAM1a-Flag were costained with a mouse anti-FLAG MAb and a rabbit anti-SADS-CoV-N pAb. Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 594-conjugated anti-rabbit IgG were costained for secondary antibody detection, followed by DAPI incubation. For challenged cells transfected with hACE2-GFP, anti-SADS-CoV-N pAb and Alexa Fluor 594-conjugated anti-rabbit IgG were used; magnification = 200×. (B) Western blot analysis also confirmed the expression of CoV receptors in transfected MDCK cells. (C) TGEV-, SARS-CoV-, MERS-CoV-, or MHV-spike-mediated pseudovirus entry into MDCK cells overexpressing the corresponding receptor. The pseudovirus entry efficiency was characterized as luciferase activity accompanying the entry. Cells transfected with the empty backbone vector were used as controls. (D) Rescue of SADS-CoV in MDCK cells transfected with a SeACoV infectious cDNA clone. Detection of expression of Nsp3-AC and N proteins of SADS-CoV was conducted at 72 h posttransfection by costaining with a rabbit anti-AC pAb and a mouse anti-N pAb (magnification = 200×). (E) Infection of fresh Vero cells with progeny SADS-CoV rescued in MDCK cells. The expression of SADS-CoV N protein was detected by staining with anti-N pAb at 36 hpi.
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