Bombyx mori nucleopolyhedrovirus ORF79 is a per os infectivity factor associated with the PIF complex

Dong, Zhanqi; Zhang, Jun; He, Qian; Cao, Mengji; Wang, La; Li, Haiqing; Xiao, Wen‐Fu; Pan, Cai-Xia; Lü, Cheng; Pan, Min‐Hui

doi:10.1016/j.virusres.2014.02.009

Cited by 35 publications

(37 citation statements)

References 39 publications

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“…We detected mRNA level of BmC‐LZM by RT‐PCR and we used the silkworm translation initiation factor 4A ( sw22934 ) gene as an internal control. To examine viral DNA replication, we performed qPCR as previously described (Dong et al ., , ; Zhang et al ., ). The gp41 , viral gene, was used to quantify viral DNA load and we performed absolute quantification with a standard to detect gp41 copies.…”

Section: Methodsmentioning

confidence: 99%

Specific genes related to nucleopolyhedrovirus in Bombyx mori susceptible and near‐isogenic resistant strains through comparative transcriptome analysis

Chen

Tan

et al. 2019

Insect Molecular Biology

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Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens that causes severe economic losses to sericulture. Comparative transcriptomics analysis has been widely applied to explore the antiviral mechanism in resistant strains. Here, to identify genes involved in BmNPV infection, we identified differentially expressed genes (DEGs) and performed weighted gene co‐expression network analysis (WGCNA) between two Bombyx mori strains: strain 871 (susceptible to BmNPV infection) and the near‐isogenic strain 871C (resistant to BmNPV). Our results showed that 400 genes were associated with resistance in strain 871C, and 76 genes were related to susceptibility in strain 871. In addition, the correlation analysis of DEGs and WGCNA showed that 40 genes related to resistance were highly expressed in the resistant strain. Among them, gene BGIBMGA004291 was the most noticeable. We further identified the effect of gene BGIBMGA004291, which encoded a multiprotein bridge factor 2 (MBF2) family member (MBF2‐10), on viral infection in cells. Our data suggested that MBF2‐10 inhibited viral infection. Taken together, this study showed specific module trait correlations related to viral infection in strains 871 and 871C, and we identified a resistance‐related gene. These findings suggested promising candidate genes with antiviral activity, aiding in the analysis of the antiviral molecular mechanisms in resistant strains.

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Section: Methodsmentioning

confidence: 99%

Specific genes related to nucleopolyhedrovirus in Bombyx mori susceptible and near‐isogenic resistant strains through comparative transcriptome analysis

Chen

Tan

et al. 2019

Insect Molecular Biology

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“…Occlusion-derived virus (OB) amplification was performed using oral inoculation with the wild-type (WT) Chongqing strain of BmNPV in silkworm larvae. OBs were harvested from the infected hemolymph before the larvae died [34].…”

Section: Methodsmentioning

confidence: 99%

“…Total DNA was extracted from silkworm cells and larvae using a Wizard Genomic DNA extraction kit (Promega, USA). The copy number of BmNPV was calculated based on quantitative PCR as previously described [34]. PCR was performed in 15 μl reactions using 1 μl of extracted DNA solution as the template.…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cpf1 mediated genome editing enhancesBombyx moriresistance to BmNPV

Dong

et al. 2020

Preprint

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18CRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new 19 opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only 20 AsCpf1 have been developed for insect genome editing, and the novel Cas12a orthologs 21 nucleases and editing efficiency require more study in insect. We compared three 22 Cas12a orthologs nucleases, AsCpf1, FnCpf1, and LbCpf1, for their editing efficiencies 23 and antiviral abilities in vitro. The three Cpf1 efficiently edited the BmNPV genome 24 and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the 25 FnCpf1 system was more efficient than the SpCas9 system after infection by BmNPV. 26 We created FnCpf1×gIE1 and SpCas9×sgIE1 transgenic hybrid lines and evaluated the 27 gene editing efficiency of different systems at the same target site. We improved the 28 antiviral ability using the FnCpf1 system in transgenic silkworm. This study 29 demonstrated use of the CRISPR/Cpf1 system to achieve high editing efficiencies in 30 the silkworm, and illustrates the use of this technology for increasing disease resistance. 31Genome editing is a powerful tool that has been widely used in gene function, gene 36 therapy, pest control, and disease-resistant engineering in most parts of pathogens 37 research. Since the establishment of CRISPR/Cas9, powerful strategies for antiviral 38 therapy of transgenic silkworm have emerged. Nevertheless, there is still room to 39 expand the scope of genome editing tool for further application to improve antiviral 40 research. Here, we demonstrate that three Cpf1 endonuclease can be used efficiency 41 editing BmNPV genome in vitro and in vivo for the first time. More importantly, this 42 Cpf1 system could improve the resistance of transgenic silkworms to BmNPV compare 43 with Cas9 system, and no significant cocoons difference was observed between 44 transgenic lines infected with BmNPV and control. These broaden the range of 45 application of CRISPR for novel genome editing methods in silkworm and also enable 46 sheds light on antiviral therapy.47 49 Genome editing introduces DNA mutations in the form of insertions, deletions or 50 base substitutions within selected DNA sequences [1]. Clustered regularly interspaced 51 short palindromic repeats (CRISPR) gene editing technology has been used in gene 52 function research, genetic improvement, modelling biology and gene therapy [2-5]. 53 Three effector proteins of class 2 type V CRISPR systems, the CRISPR/CRISPR-54 associated 12a (Cas12a, known as Cpf1) proteins of Lachnosperaceae bacterium 55 (LbCpf1), Francisella novicida (FnCpf1) and Acidaminoccocus sp. (AsCpf1), have 56 been shown to efficiently edit mammalian cell genomes with more efficient genome 57 editing than the widely used Streptococcus pyogenes Cas9 (SpCas9) [6-8]. However, 58 the CRISPR/Cpf1 system was rarely used for insect genome editing research and 59 antiviral therapy [9].60 The silk industry (Bombyx mori, B. mori), suffers great economic losses due to B. 61 mori nucleopolyhedr...

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“…The silkworm larvae and transgenic lines were reared on fresh mulberry leaves under standard conditions. The OBs were harvested from the infected larvae by gradient centrifugation (Dong et al , ). The OBs were counted using a Nageotte haemocytometer and stored at 4 °C.…”

Section: Experimetal Proceduresmentioning

confidence: 99%

CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

Dong

et al. 2018

Insect Molecular Biology

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The CRISPR/Cas9 system is a powerful tool for the treatment of infectious diseases. In our previous study, we knocked out the Bombyx mori nucleopolyhedrovirus (BmNPV) key genes and BmNPV-dependent host factor to generate transgenic antiviral strains. To further expand the range of target genes for BmNPV and more effectively prevent and control pathogenic infections, we performed gene editing and antiviral analysis by constructing a target-directed baculovirus early transcriptional activator immediate early-0 (ie-0) and 2 (ie-2) transgenic silkworm line. We hybridized it with Cas9 transgenic line to produce a double-positive transgenic Cas9(+)/sgIE0-sgIE2(+) line that could activate the CRISPR gene editing system. We first demonstrated that the system is capable of efficiently editing target genes and resulting in fragment deletions in the BmNPV genome. Survival rate of the transgenic Cas9(+)/sgIE0-sgIE2(+) line reached 65% after inoculation with 1 × 10 occlusion bodies/larva. Molecular analysis showed that BmNPV DNA replication and viral gene expression level in the transgenic Cas9(+)/sgIE0-sgIE2(+) line were significantly inhibited compared with the control Cas9(-)/sgIE0-sgIE2(-) line. These results indicated that IE-0 and IE-2, as baculovirus early transcriptional activators, can be used as target sites for gene therapy and that multigene editing could expand the range of target sites for research to create silkworm resistance breeds.

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Bombyx mori nucleopolyhedrovirus ORF79 is a per os infectivity factor associated with the PIF complex

Cited by 35 publications

References 39 publications

Specific genes related to nucleopolyhedrovirus in Bombyx mori susceptible and near‐isogenic resistant strains through comparative transcriptome analysis

Specific genes related to nucleopolyhedrovirus in Bombyx mori susceptible and near‐isogenic resistant strains through comparative transcriptome analysis

CRISPR/Cpf1 mediated genome editing enhancesBombyx moriresistance to BmNPV

CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

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