Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology

Peng, Qi; Fang, Liurong; Ding, Zhen; Wang, Dan; Peng, Guiqing; Xiao, Shaobo

doi:10.1016/j.jviromet.2019.113772

Cited by 22 publications

(31 citation statements)

References 32 publications

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“…In contrast, we used the BAC‐based system combined with homologous recombination technology to construct a full‐length cDNA infectious clone for PDCoV. This system shows several comparative advantages over its counterparts (Jengarn et al., 2015; Li et al., 2017; Peng et al., 2020). Firstly, this vector system is capable of efficiently accommodating large foreign genes and thus is suitable for the construction of infectious cDNA clone for the viruses with large genomes.…”

Section: Discussionmentioning

confidence: 99%

A strain of porcine deltacoronavirus: Genomic characterization, pathogenicity and its full‐length cDNA infectious clone

Zhou

Zhang

et al. 2020

Transbound Emerg Dis

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As a novel enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) warrants further investigation. In this study, a Chinese PDCoV strain, designated CHN-HN-1601, was isolated from the faeces of a diarrhoeic piglet. After plaque purification, the genome was determined which shared 97.5%-99.5% nucleotide identities with 71 representative PDCoV strains available in the GenBank. The pathogenic properties of CHN-HN-1601 were evaluated using 5-day-old piglets. All inoculated piglets developed severe diarrhoea from 2 days post-infection (dpi) onwards. To our surprise, two periods of diarrhoea starting from 2 to 7 dpi and from 13 to 19 dpi were observed in affected piglets during the experiment. Faecal viral shedding of the inoculated piglets was detected by real-time RT-PCR, with viral shedding peaked at 4 and 16 dpi, respectively. At necropsy at 5 dpi, the main gross lesions included transparent, thin-walled and gas-distended intestines containing yellow watery contents.Further histopathological examinations, including haematoxylin and eosin staining, immunohistochemistry and RNAscope in situ hybridization, revealed that the virus infection caused severe villous atrophy of the small intestines, with PDCoV antigen and RNA mainly distributed in the cytoplasm of the villous epithelial cells of jejunum and ileum in piglets. The dynamic production of PDCoV-specific IgG and neutralizing antibodies in serum of the affected piglets was also assessed using a whole virus-based ELISA and an immunofluorescence assay-based neutralization test, respectively. Furthermore, a full-length cDNA infectious clone of CHN-HN-1601 was constructed using a bacterial artificial chromosome system. The rescued virus exhibited in vitro growth and pathogenic properties similar to the parental virus. Taken together, our study not only enriches the information of PDCoV, but also provides a useful reverse genetics platform for further pathogenesis exploration of the virus.

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Section: Discussionmentioning

confidence: 99%

A strain of porcine deltacoronavirus: Genomic characterization, pathogenicity and its full‐length cDNA infectious clone

Zhou

Zhang

et al. 2020

Transbound Emerg Dis

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“…Briefly, several vital elements including CMV promoter (initiating viral RNA synthesis), partial N-terminal end of PDCoV genome (nt 1–375, containing a restriction enzyme site of AvrII), C-terminal end of PDCoV genome (nt 23365–25421, containing restriction enzyme site of BamHI), a 27-residue poly (A) tail, hepatitis deltavirus (HDV) ribozyme self-cleavage site and bovine growth hormone (BGH) termination sequences (efficiently stopping the transcription) [ 41 ], were cloned into pBeloBAC11 vector through the restriction sites ApaLI and HindIII ( Figure 1 A), producing an intermediate plasmid (pBAC-M-PDCoV). PDCoV-infected LLC-PK1 cells were subjected to the total RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…To establish a rapid and sensitive anti-PDCoV drug screening platform, a Nluc gene with many preponderant features, including small size, stability, and higher bioluminescence activity was designed to replace the NS6 gene due to its nonessential requirement for normal viral replication [ 38 ]. Recombinant PDCoV expressing Nluc was constructed on the basis of CRISPR/Cas9 technology, as described previously [ 41 ]. Briefly, two specific primers (sgPDCoV-ΔNS6a and sgPDCoV-ΔNS6b) targeting the upstream (523 bp) and downstream (578 bp) sequences of the NS6 gene were designed and synthesized.…”

Section: Methodsmentioning

confidence: 99%

Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase

Fang

Zhang

Sun

et al. 2021

Viruses

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Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

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“…In the current study, K289 in PEDV nsp13 was also verified as being important for ATPase and helicase activities. We attempted to generate recombinant PEDV with a K289A mutation in nsp13 via the infectious clone of PEDV strain AJ1102 ( Peng et al, 2020 ), the recombinant PEDV with a K289A mutation could not be successfully rescued, further demonstrating that K289 is essential for nsp13 helicase activity and is pivotal for viral survival.…”

Section: Discussionmentioning

confidence: 99%

ATPase and helicase activities of porcine epidemic diarrhea virus nsp13

Ren

Ding

Fang

et al. 2021

Veterinary Microbiology

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Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology

Cited by 22 publications

References 32 publications

A strain of porcine deltacoronavirus: Genomic characterization, pathogenicity and its full‐length cDNA infectious clone

A strain of porcine deltacoronavirus: Genomic characterization, pathogenicity and its full‐length cDNA infectious clone

Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase

ATPase and helicase activities of porcine epidemic diarrhea virus nsp13

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