Development of HiBiT-Tagged Recombinant Infectious Bronchitis Coronavirus for Efficient in vitro and in vivo Viral Quantification

Liang, Xiao; Zhu, Qing; Liang, Jia Qi; Liu, Si Ying; Liu, Ding Xiang; Fung, To Sing

doi:10.3389/fmicb.2020.02100

Cited by 10 publications

(5 citation statements)

References 56 publications

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“…Here, we found that the 11-amino acid HiBiT tags used to monitor VLP production and entry can be appended to different structural proteins of SARS-CoV2, as demonstrated by the VLP-cell entry assays ( Figure 6 ). This advances the NanoBiT technology to SARS-CoV2, building from a recent report that has shown the utility of HiBiT tags on spike or membrane protein of infectious bronchitis coronavirus [ 57 ]. Using sensitive NanoBiT technology, the system isolates virus-cell entry and can thereby recognize the receptors and proteases operating as coronavirus-cell susceptibility factors ( Figure 6 ).…”

Section: Discussionmentioning

confidence: 99%

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles

Kumar

Hawkins

Kicmal

et al. 2021

Cells

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Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.

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Section: Discussionmentioning

confidence: 99%

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles

Kumar

Hawkins

Kicmal

et al. 2021

Cells

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“…In order to assess the effect of Retro-2.2 on F trafficking, we then quantified the presence of F at the plasma membrane. To overcome the potential bias linked to cell fusion and of immunostaining, we generated an F surrogate which consisted in the insertion in place of the F ectodomain of the sequences of the mCherry and of the HiBit peptide, an 11-amino-acid-long optimized peptide of the split Nano Luciferase ( Figure 3 A) [ 40 , 41 , 42 ]. The plasmid encoding this chimeric protein was transfected in HEp-2 cells which were incubated for 24 h in the absence or presence of 2 µM Retro-2.2.…”

Section: Resultsmentioning

confidence: 99%

A New Derivative of Retro-2 Displays Antiviral Activity against Respiratory Syncytial Virus

Le Rouzic,

Fix,

Vinck

et al. 2023

IJMS

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Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia in newborns, with all children being infected before the age of two. Reinfections are very common throughout life and can cause severe respiratory infections in the elderly and immunocompromised adults. Although vaccines and preventive antibodies have recently been licensed for use in specific subpopulations of patients, there is still no therapeutic treatment commonly available for these infections. Here, we investigated the potential antiviral activity of Retro-2.2, a derivative of the cellular retrograde transport inhibitor Retro-2, against hRSV. We show that Retro-2.2 inhibits hRSV replication in cell culture and impairs the ability of hRSV to form syncytia. Our results suggest that Retro-2.2 treatment affects virus spread by disrupting the trafficking of the viral de novo synthetized F and G glycoproteins to the plasma membrane, leading to a defect in virion morphogenesis. Taken together, our data show that targeting intracellular transport may be an effective strategy against hRSV infection.

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“…Recovery of rIBV-Beau-KC(S1) from the cloned cDNA was performed according to a protocol that was successfully used for the recovery of a HiBiT-tagged recombinant IBV (Liang et al, 2020). Vero cells were electroporated with the in vitro transcribed full-length and N transcripts and cultured up to 96 h post-electroporation.…”

Section: Resultsmentioning

confidence: 99%

Establishment and Cross-Protection Efficacy of a Recombinant Avian Gammacoronavirus Infectious Bronchitis Virus Harboring a Chimeric S1 Subunit

et al. 2022

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Infectious bronchitis virus (IBV) is a gammacoronavirus that causes a highly contagious disease in chickens and seriously endangers the poultry industry. A diversity of serotypes and genotypes of IBV have been identified worldwide, and the currently available vaccines do not cross-protect. In the present study, an efficient reverse genetics technology based on Beaudette-p65 has been used to construct a recombinant IBV, rIBV-Beaudette-KC(S1), by replacing the nucleotides 21,704–22,411 with the corresponding sequence from an isolate of QX-like genotype KC strain. Continuous passage of this recombinant virus in chicken embryos resulted in the accumulation of two point mutations (G21556C and C22077T) in the S1 region. Further studies showed that the T248S (G21556C) substitution may be essential for the adaptation of the recombinant virus to cell culture. Immunization of chicks with the recombinant IBV elicited strong antibody responses and showed high cross-protection against challenges with virulent M41 and a QX-like genotype IBV. This study reveals the potential of developing rIBV-Beau-KC(S1) as a cell-based vaccine with a broad protective immunity against two different genotypes of IBV.

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Development of HiBiT-Tagged Recombinant Infectious Bronchitis Coronavirus for Efficient in vitro and in vivo Viral Quantification

Cited by 10 publications

References 56 publications

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles

A New Derivative of Retro-2 Displays Antiviral Activity against Respiratory Syncytial Virus

Establishment and Cross-Protection Efficacy of a Recombinant Avian Gammacoronavirus Infectious Bronchitis Virus Harboring a Chimeric S1 Subunit

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