Establishment of replication-competent vesicular stomatitis virus recapitulating SADS-CoV entry

Abstract

Zoonotic coronaviruses pose a continuous threat to human health, with newly identified bat-borne viruses like swine acute diarrhea syndrome coronavirus (SADS-CoV) causing high mortality in piglets. In vitro studies indicate that SADS-CoV can infect cell lines from diverse species, including humans, highlighting its potential risk to human health. However, the lack of tools to study viral entry, along with the absence of vaccines or antiviral therapies, perpetuates this threat. To address this, we engineered an infectious molecular clone of Vesicular Stomatitis Virus (VSV), replacing its native glycoprotein (G) with SADS-CoV spike (S) and inserting a Venus reporter at the 3' leader region to generate a replication-competent rVSV-Venus-SADS S virus. Serial passages of rVSV-Venus-SADS S led to the identification of an 11-amino-acid truncation in the cytoplasmic tail of the S protein, which allowed more efficient viral propagation due to increased cell membrane anchoring of the S protein. The S protein was integrated into rVSV-Venus-SADS SΔ11 particles, susceptible to neutralization by sera from SADS-CoV S1 protein-immunized rabbits. Additionally, we found that TMPRSS2 promotes SADS-CoV spike-mediated cell entry. Furthermore, we assessed the serum-neutralizing ability of mice vaccinated with rVSV-Venus-SADS SΔ11 using a prime-boost immunization strategy, revealing effective neutralizing antibodies against SADS-CoV infection. In conclusion, we have developed a safe and practical tool for studying SADS-CoV entry and exploring the potential of a recombinant VSV-vectored SADS-CoV vaccine.IMPORTANCEZoonotic coronaviruses, like swine acute diarrhea syndrome coronavirus (SADS-CoV), pose a continual threat to human and animal health. To combat this, we engineered a safe and efficient tool by modifying the Vesicular Stomatitis Virus (VSV), creating a replication-competent rVSV-Venus-SADS S virus. Through serial passages, we optimized the virus for enhanced membrane anchoring, a key factor in viral propagation. This modified virus, rVSV-Venus-SADS SΔ11, proved susceptible to neutralization, opening avenues for potential vaccines. Additionally, our study revealed the role of TMPRSS2 in SADS-CoV entry. Mice vaccinated with rVSV-Venus-SADS SΔ11 developed potent neutralizing antibodies against SADS-CoV. In conclusion, our work presents a secure and practical tool for studying SADS-CoV entry and explores the promise of a recombinant VSV-vectored SADS-CoV vaccine.

Keywords: SADS-CoV; VSV; VSV-vectored vaccine; replication-competent; viral entry.

Fig 1

Recovery and adaptation of the replication-competent rVSV-Venus-SADS S virus. (A) Schematic diagram illustrating the genome organization in the pVSV-Venus-SADS S plasmid. The complete SADS-CoV spike replaces the native VSV G and Venus is inserted into the first position of the genome. Plasmid transcription is driven by the T7 promoter and terminated by the T7 terminator. A hepatitis delta virus ribozyme sequence is included for the generation of the authentic 5′ end of the viral RNA genome. (B) Schematic representation of the generation process of rVSV-Venus-SADS S. The pVSV-Venus-SADS S plasmid, expressing T7 RNA polymerase plasmids, and helper plasmids were transfected into HEK293T cells to produce P0 virus (see Materials and Methods). This was followed by infection of Huh7.5.1 cells with the cell culture supernatant from the HEK293T cells. (C) Serial passages of the supernatant and virus-infected cells were performed every 3 or 4 days. Venus signals were observed under the microscope. (D) The morphology of the plaques from P2 and P6 virus was assessed in Huh7.5.1 cells monolayer 4 days post-infection (dpi). (E) Measurements of plaque diameter were conducted using ImageJ software. (F) Total RNAs were extracted and real-time quantitative PCR (RT-qPCR) assays were conducted to determine viral RNA levels. Error bars represent the standard deviations from one of two independent experiments performed in triplicate. Statistical analysis was performed using an unpaired two-tailed t-test. ***, P < 0.001; ****, P < 0.0001.

Fig 2

Identification of S gene mutations facilitating chimeric virus infection. (A) RT-PCR of P2 and P6 virus-infected cells and sequencing analysis of spike gene mutations during serial passages. (B) Reverse genetics engineered the S and SΔ11 genes on the VSV genome and the two viruses were rescued simultaneously. The growth kinetics of the two viruses was characterized in Huh7.5.1 cells. The dashed line indicates the detection limit. Error bars represent the standard deviations from one of two independent experiments performed in triplicate. (C) Plaque phenotypes of rVSV-Venus-SADS S and rVSV-Venus-SADS SΔ11 virus were assessed in Huh7.5.1 cells monolayer 4 dpi. (D) Measurements of plaque diameter were made using ImageJ software. Statistical analysis was performed by an unpaired two-tailed t-test. ****, P < 0.0001. (E) Observation of the fluorescence signal of the two viruses de novo infected Huh7.5.1 cells.

Fig 3

Sequences in the cytoplasmic tail of the SADS-CoV spike modulate its cell surface localization and syncytia formation. (A) Display of sequences in the cytoplasmic tail of WT S and SΔ11. (B) The schematic diagram for detecting spike protein subcellular distribution. Briefly, 6-well plates of 293T cells were transfected with 4 µg pCAG-SADS S and pCAG-SADS SΔ11 plasmids, respectively. Twenty-four hours post-transfection, the cells were divided into two parts. One part, detecting spikes on the cell surface, was initially stained under nonpermeabilizing conditions, and the other part, detecting spikes on the whole cell, was permeabilized and stained with rVSV-Venus-SADS SΔ11 immunized mouse sera. (C) Analysis of spike distribution on the cell surface and in the whole cells using Flow cytometry. (D) The quantitative ratio of WT S and mutant SΔ11 anchored to the cell surface, respectively. Results are representative of at least three independent experiments. Statistical analysis was performed by unpaired two-tailed t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) Schematic representation of syncytium formation between donor cells expressing spike protein and split GFP, and acceptor cells expressing split GFP. (F) The images were taken at 6 h and 24 h by Nikon Ti2-U, with at least three fields for each condition. Scale bar, 100 µm. (G) Measurement of GFP-positive area and fused cell number, and the average GFP areas of syncytia measured by ImageJ. Results are mean ± SD from at least three fields per condition.

Fig 4

Characterization of the infectious rVSV-Venus-SADS SΔ11 chimera. (A) Purified rVSV-Venus-SADS SΔ11 particles were examined by negative stain electron microscopy; scale bars represent 250 nm. (B) Infectious rVSV-Venus-SADS SΔ11 particles were purified through continuous 10% to 60% sucrose gradient centrifugation. The infectivity, RNA levels, and SADS-CoV spike abundance of each fraction were assessed by titration, RT-qPCR, and Western blot, respectively. The results were expressed as FFU per mL and viral RNA genome copies per μg, respectively. Spike protein levels were detected using rVSV-Venus-SADS SΔ11 immunized mouse sera. (C) Neutralization assays were conducted with SADS-CoV S1 pAb (rabbit) against rVSV-Venus-SADS SΔ11 and rVSV-Venus-SARS-CoV-2 S. The infection experiments were performed in triplicates. Error bars represent the standard deviations from one of two independent experiments performed in triplicate.

Fig 5

TMPRSS2 facilitates SADS-CoV infection. (A) Western blot analysis of lentivirus-transduced cell lines expressing HA-TMPRSS2. (B) rVSV-Venus-SADS SΔ11 infection in different cell lines at MOI 0.3. Fluorescence was observed at 24 hpi using a Nikon Ti2-U microscope. Scale bar, 500 µm. (C) Flow cytometry analysis of rVSV-Venus-SADS SΔ11 infection in different cell lines. (D and E) Huh7.5.1 and Huh7.5.1 cells overexpressing TMPRSS2 were infected with rVSV-Venus-SADS SΔ11 and authentic SADS-CoV, respectively, with the indicated camostat doses, and infection was subsequently scored. All infections were performed in triplicate, and the data represent three independent experiments (mean ± SD).

Fig 6

Prime-Boost vaccination of C57BL/6 mice elicits neutralizing antibodies against SADS-CoV. (A) Prime-boost vaccination schedule. C57BL/6 female mice (n = 7) were initially immunized with rVSV-Venus-SADS SΔ11 and subsequently boosted with homologous viruses 2 weeks after the primary vaccination. Sera were collected on day 13, one day before primary vaccination, and day 28, 2 weeks after booster vaccination. (B) Body weight changes in the PBS-injected group compared to the rVSV-Venus-SADS SΔ11 vaccinated group. (C) Determination of neutralizing antibody levels in the vaccinated mice group against rVSV-Venus-SADS SΔ11 infection quantified as the GFP positive rate and normalized to the virus without sera. A sigmoidal dose–response curve was fitted to the data using Prism GraphPad 8.0 (GraphPad Software). The GFP reduction 50% neutralizing titer (NT₅₀) of pre-, prime-, and boost-vaccination mouse sera were compared by P-value (paired t-test). The dashed line indicates the detection limit. The values listed above represent the average NT₅₀ of pre-, prime-, and boost-vaccination sera. (D) Determined the neutralizing antibody level in five boost-vaccination mice against SADS-CoV infection, quantified by SADS-CoV N protein expression through immunostaining and normalized to the virus without sera. The 50% neutralizing titer (NT₅₀) of the five boost-vaccination mouse sera was evaluated.