A highly attenuated SARS-CoV-2 related pangolin coronavirus variant has a 104nt deletion at the 3′-terminus untranslated region

  1. Shanshan Lu
  2. Shengdong Luo
  3. Chang Liu
  4. Muli Li
  5. Xiaoping An
  6. Mengzhe Li
  7. Jun Hou
  8. Huahao Fan
  9. Panyong Mao
  10. Yigang Tong
  11. Lihua Song

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Abstract

SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3’-terminus untranslated region (3’-UTR). We further report the characterization of the GX_P2V variant in in vitro and in vivo infection models. In cultured Vero and BGM cells, the GX_P2V variant produced minimal cell damage and small plaques. The GX_P2V variant infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V variant is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely due to the 104-nt deletion in the HVR in the 3’-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2022.02.07.479352: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsEuthanasia Agents: For intranasal infection, hamsters were infected with 104 or 105 TCID50 of GX_P2V in 50 μL RPMI 1640 containing 2% FBS after deep anesthesia with pentobarbital sodium.
    Sex as a biological variableGolden hamster model of both intranasal and intragastrical infection: Specific pathogen-free, 6-week-old male golden hamsters were purchased from Charles River and maintained under specific-pathogen-free conditions.
    RandomizationYoung and old mice were randomly divided into two groups, respectively.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Sera were collected at 42 d.p.i for analysis of antibody responses by immunofluorescence assay (IFA), ELISA, and pseudovirus neutralization titer assay (pVNT).

    Table 2: Resources

    Antibodies
    SentencesResources
    After being washed five times with PBS, cells were stained with secondary antibodies: fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (1:300, ZSGB-BIO, Cat No. ZF-0312, Beijing, China) or goat anti-human IgG (1:300, ZSGB-BIO, Cat No. ZF-0308, Beijing, China), accordingly.
    anti-mouse IgG
    suggested: (ZSGB-Bio Cat# ZF-0312, RRID:AB_2716306)
    anti-human IgG
    suggested: None
    ELISA binding assay: SARS-CoV-2 RBD, S1 and S1+S2 (ECD) antibody titer assay kits (Sino Biological, Cat No. KIT002, KIT003, KIT004, respectively, Beijing, China) were purchased.
    ECD
    suggested: None
    KIT003
    suggested: None
    KIT004 , respectively , Beijing , China
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viral growth kinetics: Confluent monolayers of BGM and Vero cells in 6-well plates were incubated with viral stocks at a multiplicity of infection (MOI) of 0.01 for 2 h at 37°C followed by washing twice with PBS and maintained in RMPI 1640 medium with 2% FBS and HEPES (20 mM) at 37°C in a 5% CO2 incubator.
    Vero
    suggested: None
    Confluent monolayers of 293T cells grown on coverslips in 12-well plates overnight were transfected with plasmids containing codon-optimized spike genes of SARS-CoV-2 and GX_P2V by using liposome (TransGen, Cat No. FT201-02, Beijing, China) according to the instructions.
    293T
    suggested: None
    The SARS-CoV-2 pseudovirus was used as seed stock and combined with the GX_P2V spike protein expression plasmid to generate VSV-based GX_P2V pseudovirus in 293T-hACE2 cells.
    293T-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    For testing sera from hamsters and BALB/c mice, the secondary antibodies were replaced by HRP-goat anti-mouse IgG (1:5000, ZSGB-BIO).
    BALB/c
    suggested: None
    Recombinant DNA
    SentencesResources
    In addition, a standard plasmid was constructed through inserting a fragment amplified using P2VF and P2VR into a cloning vector pEASY-T1 (TransGen, Cat No. CT101-01 Beijing, China)
    pEASY-T1
    suggested: None
    Immunofluorescence assay for cross-immunity responses: The full-length spike protein gene of SARS-CoV-2 (accession number NC_045512.2) and GX_P2V (accession number MW532698) were codon optimized, synthesized, and cloned into pcDNA3.1 vector by Beijing BioMed Gene Technology.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    Software and Algorithms
    SentencesResources
    The clean reads were then mapped to the near complete genome of pangolin coronavirus GX_P2V (GenBank accession number MT072864) using Bowtie 231.
    Bowtie
    suggested: (Bowtie, RRID:SCR_005476)
    The reads that were mapped to the GX_P2V genome were then assembled de novo using Trinity with default settings32.
    Trinity
    suggested: (Trinity, RRID:SCR_013048)
    Statistical analysis: The statistical analyses were performed with GraphPad Prism version 9.0 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2022.02.07.479352 on bioRxiv