Replication of MERS and SARS coronaviruses in bat cells offers insights to their ancestral origins

Abstract

Previous findings of Middle East Respiratory Syndrome coronavirus (MERS-CoV)-related viruses in bats, and the ability of Tylonycteris-BatCoV HKU4 spike protein to utilize MERS-CoV receptor, human dipeptidyl peptidase 4 hDPP4, suggest a bat ancestral origin of MERS-CoV. We developed 12 primary bat cell lines from seven bat species, including Tylonycteris pachypus, Pipistrellus abramus and Rhinolophus sinicus (hosts of Tylonycteris-BatCoV HKU4, Pipistrellus-BatCoV HKU5, and SARS-related-CoV respectively), and tested their susceptibilities to MERS-CoVs, SARS-CoV, and human coronavirus 229E (HCoV-229E). Five cell lines, including P. abramus and R. sinicus but not T. pachypus cells, were susceptible to human MERS-CoV EMC/2012. However, three tested camel MERS-CoV strains showed different infectivities, with only two strains capable of infecting three and one cell lines respectively. SARS-CoV can only replicate in R. sinicus cells, while HCoV-229E cannot replicate in any bat cells. Bat dipeptidyl peptidase 4 (DPP4) sequences were closely related to those of human and non-human primates but distinct from dromedary DPP4 sequence. Critical residues for binding to MERS-CoV spike protein were mostly conserved in bat DPP4. DPP4 was expressed in the five bat cells susceptible to MERS-CoV, with significantly higher mRNA expression levels than those in non-susceptible cells (P = 0.0174), supporting that DPP4 expression is critical for MERS-CoV infection in bats. However, overexpression of T. pachypus DPP4 failed to confer MERS-CoV susceptibility in T. pachypus cells, suggesting other cellular factors in determining viral replication. The broad cellular tropism of MERS-CoV should prompt further exploration of host diversity of related viruses to identify its ancestral origin.

Fig. 1. The twelve bat cell lines and Vero cells were subject to infection by MERS-CoV in clade A and clade B.

The 12 bat cell lines (PAK: Pipistrellus abramus kidney, PAL Pipistrellus abramus lung, RSK: Rhinolophus sinicus kidney, RSL: Rhinolophus sinicus lung, MRK: Myotis ricketti kidney, MRL: Myotis ricketti lung, TPK: Tylonycteris pachypus kidney, TPL: Tylonycteris pachypus lung, HPK: Hipposideros pomona kidney, RLK: Rousettus leschenaultii kidney, RLL: Rousettus leschenaultii lung, MPK: Miniopterus pusillus kidney) and Vero cells were subject to infection by MERS-CoV EMC/2012 (belonging to clade A1) with MOI of 1 (a). Culture supernatants were harvested at day 0 and 5 postinfection. Viral titers were determined by real-time quantitative RT-PCR. Viral load was expressed as log₁₀ copies/mL. Error bars indicate the standard deviation of triplicate samples. The five bat cell lines susceptible to MERS-CoV EMC/2012 infection with ≥ 1 log₁₀ increase in viral load at day 5 were marked with red triangles. They were subject to infection by three other MERS-CoV strains isolated from camels in Dubai, D998/15 (belonging to clade A2) (b), D1189.1/15 (c) and D1271.1/15 (d) (belonging to clade B3) (b). Different MERS-CoV strains displayed different infectivities in these five bat cells. (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 2. Cytopathic effects (CPE) were observed in infected M. ricketti lung, R. sinicus lung and Vero cells on 5 days postinfection.

CPE was compared between Myotis ricketti lung (immortalized) cells that were uninfected (control) (a), and infected with Dubai camel MERS strains D998/15 (b), D1189.1/15 (c) & D1271.1/15 (d). CPE was compared between Rhinolophus sinicus lung (immortalized) cells that were uninfected (control) (e), and infected with Dubai camel MERS strains D998/15 (f), D1189.1/15 (g), and D1271.1/15 (h). CPE was compared between Vero cells that were uninfected (control) (i), and infected with Dubai camel MERS strains D998/15 (g), D1189.1/15 (k), and D1271.1/15 (l)

Fig. 3. The twelve bat cell lines and Vero/HFL cells were subject to infection by SARS-CoV and HCoV-229E.

The 12 bat cell lines (PAK: Pipistrellus abramus kidney, PAL Pipistrellus abramus lung, RSK: Rhinolophus sinicus kidney, RSL: Rhinolophus sinicus lung, MRK: Myotis ricketti kidney, MRL: Myotis ricketti lung, TPK: Tylonycteris pachypus kidney, TPL: Tylonycteris pachypus lung, HPK: Hipposideros pomona kidney, RLK: Rousettus leschenaultii kidney, RLL: Rousettus leschenaultii lung, MPK: Miniopterus pusillus kidney) and Vero/HFL cells were subject to infection by SARS-CoV with MOI of 1 (a) and HCoV-229E with MOI of 0.01(b). Culture supernatants were harvested at day 0 and 5 postinfection. Viral titers were determined by real-time quantitative RT-PCR. Viral load was expressed as log₁₀ copies/mL. Error bars indicate the standard deviation of triplicate samples. Only RSK cells can support SARS-CoV infection with ≥ 1 log₁₀ increase in viral load at day 5 (blue triangle) and none of the 12 bat cell lines support HCoV-229E infection. (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 4. Phylogenetic analyses of partial DPP4 mRNA sequences and comparison of critical amino acid residues of human, camels, bats and other animals.

(a) The trees were constructed by Neighbor-Joining method using JTT substitution models and bootstrap values calculated from 1000 trees. Only bootstrap values >70% are shown. One hundred and twelve aa positions were included in the analyses. The scale bars represent 20 substitutions per site. Bat DPP4s that are sequenced in this study are labeled with black circles. Comparison of critical amino acid residues in DPP4 from different animal host for receptor binding in the region of residues 229–346 with respect to human DPP4 (b)

Fig. 5. DPP4 expression analysis of bat cell lines.

(a) mRNA levels of DPP4 were measured in various bat cells extracts by RT-qPCR and plotted relative to Vero cells, normalized by β-actin mRNA levels. The mRNA levels of DPP4 were compared between susceptible and non-susceptible bat cell lines (b). Statistical significance was assessed by Student’s t test P < 0.05

Fig. 6. Infection assay of T. pachypus lung and kidney cells with tpDPP4 overexpression.

Cells were infected with MERS-CoV at a multiplicity of infection (MOI) of 1 for 5 days. Determination of MERS-CoV viral load in supernatant (n = 3) by RT-qPCR with normalization to β-actin (represented by bar). Determination of tpDPP4 expression in cell lysates (n = 3) by RT-qPCR with normalization to beta-actin (represented by dot). (*P < 0.05, **P < 0.01, ***P < 0.001)