Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice

Abstract

Defining prospective pathways by which zoonoses evolve and emerge as human pathogens is critical for anticipating and controlling both natural and deliberate pandemics. However, predicting tenable pathways of animal-to-human movement has been hindered by challenges in identifying reservoir species, cultivating zoonotic organisms in culture, and isolating full-length genomes for cloning and genetic studies. The ability to design and recover pathogens reconstituted from synthesized cDNAs has the potential to overcome these obstacles by allowing studies of replication and pathogenesis without identification of reservoir species or cultivation of primary isolates. Here, we report the design, synthesis, and recovery of the largest synthetic replicating life form, a 29.7-kb bat severe acute respiratory syndrome (SARS)-like coronavirus (Bat-SCoV), a likely progenitor to the SARS-CoV epidemic. To test a possible route of emergence from the noncultivable Bat-SCoV to human SARS-CoV, we designed a consensus Bat-SCoV genome and replaced the Bat-SCoV Spike receptor-binding domain (RBD) with the SARS-CoV RBD (Bat-SRBD). Bat-SRBD was infectious in cell culture and in mice and was efficiently neutralized by antibodies specific for both bat and human CoV Spike proteins. Rational design, synthesis, and recovery of hypothetical recombinant viruses can be used to investigate mechanisms of transspecies movement of zoonoses and has great potential to aid in rapid public health responses to known or predicted emerging microbial threats.

Fig. 1.

Schematic representation of SARS-CoV and Bat-SCoV variants. (A) Schematic representation of SARS-CoV and Bat-SCoV (GenBank accession no. FJ211859) genomes and reverse genetics system. (Top) Arrowheads indicate nsp processing sites within the ORF1ab polyprotein (open arrowheads, papain-like proteinase mediated; filled arrowheads, nsp5 [3C-like proteinase] mediated). Immediately below are the fragments used in the reverse genetics system, labeled A through F. The fragments synthesized to generate Bat-SCoV exactly recapitulate the fragment junctions of SARS-CoV with the exception that the Bat-SCoV has 2 fragments, Bat-E1 and Bat-E2, which correspond to the SARS-E fragment. (B) Schematic representation showing organization of the SARS-CoV and Bat-SCoV Spike proteins. The engineered Spike proteins are pictured below with the virus name to the left. Bat-SRBD includes all of the Bat-SCoV Spike sequence except that the Bat-SCoV RBD (Bat-SCoV amino acid 323–505) is replaced with the SARS-CoV RBD (amino acid 319–518) (GenBank accession no. FJ211860). Bat-SRBD-MA includes the MA15 Spike RBD change at SARS-CoV aa Y436H. Bat-SRBM includes the minimal 13 SARS-CoV residues critical for ACE2 contact, resulting in a chimeric RBD of Bat-SCoV amino acid 323I-429T and SARS-CoV amino acid 426R-518D. Bat-Hinge is Bat-SRBM sequence, with Bat-SCoV amino acid 392L-397E replaced with SARS-CoV amino acid 388V-393D. Bat-F includes nt 1–24057 of SARS-CoV (to Spike amino acid 855), with the remaining 3′ sequence from Bat-SCoV. To the right of the schematic representations, observation of transcript activity and approximate stock titers at passage 1 (P1) are indicated. ND indicates no infectious virus detected by plaque assay. (C and D) Presence of genomic and subgenomic transcripts after electroporation of in vitro transcribed viral RNA. Band corresponding to mRNA1 indicates the presence of genomic RNA, either electroporated genomic RNA or progeny genomic RNA, and the presence of a band corresponding to mRNA9 indicates the presence of leader-containing subgenomic RNA, consistent with mRNA transcription.

Fig. 2.

Growth of SARS-CoV and Bat-SRBD in 3 different cell types. (A–C) Vero cells (A), DBT-hACE2 cells (B), or DBT-cACE2 cells (C) were infected with SARS-CoV at a MOI = 1 (■) or MOI = 0.01 (●), or Bat-SRBD at a MOI = 1 (□) or a MOI = 0.01 (○). Infected cultures were sampled, in triplicate, at the times indicated and viral titer was quantified by plaque assay on Vero cells. Error bars indicate SD.

Fig. 3.

Neutralization of the Bat-SRBD by mouse serum and human mAbs. (A) Immune sera from 5 mice (1, ○; 2, ▾; 3, ×; 4, ▵; and 5, *) vaccinated with Bat-SCoV Spike were used to neutralize Bat-SRBD. Controls include prebleed serum (■) and Mouse 1 serum used to neutralize SARS-CoV (♦). (B) Human mAbs S109.8 (■), S227.14 (▴), and S230.15 (○) were used to neutralize Bat-SRBD. Results are expressed as the percentage of neutralization. Error bars indicate SD.

Fig. 4.

Efficient replication of Bat-SRBD in human ciliated airway epithelial cells. (A) Growth curves for SARS-CoV and Bat-SRBD were obtained from apical washes of human ciliated airway epithelial cell cultures inoculated with either virus. Samples were serially diluted and titers determined by plaque assay on Vero cells. Titers are expressed as PFU per mL. Both SARS-CoV and Bat-SRBD replicated to titers of ≈10⁷, although Bat-SRBD growth was delayed compared with SARS-CoV. All inoculations were performed in duplicate. SARS-CoV, ●; Bat-SRBD, ○. (B–D) Representative histological sections of HAE 144 h p.i. with SARS-CoV (B), Bat-SRBD (C), or vehicle alone (D) and probed with mouse polyclonal sera directed against the Bat-CoV Spike and visualized with mouse-specific secondary antibodies conjugated to AlexaFluor 488 (green). Detection of Spike immunoreactivity was localized specifically to the apical surface of ciliated cells indicating that SARS-CoV and Bat-SRBD both infect ciliated cells after apical inoculation. Note that at 144 h p.i. cilial morphology shows considerable cytotoxicity. Spike immunoreactivity was not observed in nonciliated cell-types. (Scale bar, 5 μm.)

Fig. 5.

Weight loss and viral replication of Bat-SRBD, Bat-SRBD-MA, and SARS-CoV in aged BALB/c mice. Ten 14-month-old female BALB/c mice were infected intranasally with 10⁵ PFU of the indicated virus or an equivalent volume (50 μL) of PBS. (A) Weights of all surviving mice per infection group were recorded each day, averaged, and plotted as a percentage of starting weight. Error bars indicate SD. (B) On days 2 and 4 p.i., 5 mice per group were killed and lungs were harvested. Lung homogenates were titered on Vero cells. Circles represent titers of individual mouse lungs. Bars represent the average titer of each infection group. BSRBD, Bat-SRBD; BSRM, Bat-SRBD-MA; SARS, SARS-CoV.