Viral Quantitative Capillary Electrophoresis for Counting and Quality Control of RNA Viruses

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• 作者：Afnan Azizi ; Gleb G. Mironov ; Darija Muharemagic ; Mohamed Wehbe ; John C. Bell ; Maxim V. Berezovski
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：November 6, 2012
• 年：2012
• 卷：84
• 期：21
• 页码：9585-9591
• 全文大小：306K
• 年卷期：v.84,no.21(November 6, 2012)
• ISSN：1520-6882

文摘

The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze鈥搕haw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10⁸ to 10¹³ ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation.