Establishment of EvaGreen qPCR for detecting bovine rotavirus based on VP7 gene

Wei, Shaozhong; Che, Tuanjie; Wang, Jingming; Yukong, Li; Zhang, Taojie; Changjun, Song; Tian, Fei

doi:10.5897/ajmr2013.5953

Cited by 2 publications

(2 citation statements)

References 29 publications

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“…Total RNAs were extracted from bovine coronavirus (BCoV) using the TIANamp Virus DNA/RNA Kit in accordance with the manufacturer’s standard protocol and reversely transcribed into cDNA using the M-MLV reverse transcriptase (Invitrogen) system according to the manufacture's protocols (Wei et al 2014 , 2017 ). Real-time polymerase chain reaction (PCR) was conducted with SYBR Green PCR Master Mix (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Bovine coronavirus nucleocapsid suppresses IFN-β production by inhibiting RIG-I-like receptors pathway in host cells

et al. 2022

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The present study aimed to explore if bovine coronavirus nucleocapsid (BCoV N) impacts IFN-β production in the host cells and to reveal further molecular mechanism of BCoV pathogenesis. Human embryonic kidney (HEK) 293 T cells were transiently transfected with pMyc-BCoV-N recombinant plasmids, then infected with the vesicular stomatitis virus (VSV). Expression levels of beta interferon (IFN-β) mRNA were detected using RT-qPCR. The results showed that BCoV N gene was 1347 bp that was consistent with the expected size. pMyc-BCoV-N recombinant protein was 1347 bp which was successfully transcribed and overexpressed in HEK 293 T cells. BCoV-N recombinant protein inhibited dose-dependently VSV-induced IFN-β production ( p < 0.01). MDA5, MAVS, TBK1 and IRF3 could promote transcription levels of IFN-β mRNA. But, BCoV-N protein demoted IFN-β transcription levels induced by MDA5, MAVS, TBK1 and IRF3. Furthermore, expression levels of MDA5, MAVS, TBK1 and IRF3 mRNAs were reduced in RIG-I-like receptor (RLR) pathway. In conclusion, BCoV-N reduced IFN-β levels in RIG-I-like receptor (RLR) pathway in HEK 293 T cells which were induced by MDA5, MAVS, TBK1 and IRF3(5D). BCoV-N protein inhibited IFN-β production and activation of RIG-I-like receptors ( RLRs) signal pathway. Our findings demonstrated BCoV N protein is an IFN-β antagonist through inhibition of MDA5, MAVS, TBK1 and IRF3(5D) in RLRs pathway, also revealed a new mechanism of BCoV N protein to evade host innate immune response by inhibiting type I IFN production, which is beneficial to developing novel prevention strategy for BCoV disease in the animals and humans.

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Section: Methodsmentioning

confidence: 99%

Bovine coronavirus nucleocapsid suppresses IFN-β production by inhibiting RIG-I-like receptors pathway in host cells

et al. 2022

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“…Total RNAs were extracted from BPV using the TIANamp Virus DNA/RNA Kit in accordance with the manufacturer's standard protocol and reversely transcribed into cDNA using the M-MLV reverse transcriptase (Invitrogen) system according to the manufacturer's protocols (Wei et al 2014(Wei et al , 2017.…”

Section: Construction Of Eukaryotic Expression Vector Of Bpv Vp1 Genementioning

confidence: 99%

Immune escape of bovine parvovirus by VP1 inhibiting IFN-β production through the RIG-I-like receptor pathway

Gong

Zhaofang

et al. 2023

Int Microbiol

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Objective The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-β) production and to reveal further molecular mechanism of BPV immune escape. Method The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-β mRNA were detected using qPCR. ResultsThe expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 10 4 . Expression levels of IFN-β mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-β expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. Conclusion pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-β. Overexpression of pCMV-Myc-BPV-VP decreased IFN-β mRNA expression in HEK 293 T cells and inhibited IFN-β production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.

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Establishment of EvaGreen qPCR for detecting bovine rotavirus based on VP7 gene

Cited by 2 publications

References 29 publications

Bovine coronavirus nucleocapsid suppresses IFN-β production by inhibiting RIG-I-like receptors pathway in host cells

Bovine coronavirus nucleocapsid suppresses IFN-β production by inhibiting RIG-I-like receptors pathway in host cells

Immune escape of bovine parvovirus by VP1 inhibiting IFN-β production through the RIG-I-like receptor pathway

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