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Development of a Honey Bee RNA Virus Vector Based on the Genome of a Deformed Wing Virus.
Viruses ( IF 5.818 ) Pub Date : 2020-03-28 , DOI: 10.3390/v12040374
Eugene V Ryabov 1 , Krisztina Christmon 1, 2 , Matthew C Heerman 1 , Francisco Posada-Florez 1 , Robert L Harrison 3 , Yanping Chen 1 , Jay D Evans 1
Affiliation  

We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee (Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. The in vitro RNA transcripts from egfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV with egfp inserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV leader protein (LP) and/or viral protein (VP) 2. Analysis of the progeny egfp-tagged DWV showed gradual accumulation of genome deletions for egfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.

中文翻译:

基于变形翼病毒基因组的蜜蜂RNA病毒载体的开发。

我们基于一种像picorna一样的变形翼病毒(DWV)的基因组开发了一种蜜蜂RNA病毒载体,该病毒是蜜蜂(Apis mellifera)的主要病毒病原体。为了测试DWV用作载体的潜力,将编码增强型绿色荧光蛋白(eGFP)的717 nt序列侧翼插入病毒蛋白酶靶向的肽,将其插入到DWV的感染性cDNA克隆中前导蛋白和病毒结构蛋白VP2基因。将带有egfp标签的DWV cDNA克隆的体外RNA转录物注射到蜜蜂p中后具有感染力。如氯化铯密度梯度离心所证明的,产生了稳定的DWV颗粒,其含有回收的DWV的基因组RNA和egfp插入片段。当腹腔内注射时,这些颗粒会感染蜜蜂的p。
更新日期:2020-04-20
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