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

Abstract

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-thaw 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 10(8) to 10(13) ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation.

Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

“…However, in the presence of NOGlc the infectivity decreased by only 0.2 Log 10  PFU mL −1 after 40 days storage at room temperature (Figure 2a). These results were confirmed by viral quantitative capillary electrophoresis (viral qCE)36, where the intact virus particles were separated from free DNA present after virus degradation (Figure S1). VV degraded slower when incubated with NOGlc than with PBS as more VV peaks and less DNA was observed.…”

An Improved Method on Isolation and Serial Passage ofChlamydia pneumoniaeFrom Human Peripheral Blood Mononuclear Cells

“…Hep‐2 cells infected with Cpn were broken by freeze‐thaw method twice . The broken cell suspension (concentration of Cpn was adjusted to 10 3 IFU/ml) was passed onto fresh monolayer of Hep‐2 cells in each of the five wells (0.1 ml each), and the left suspension with original concentration was added into two culture bottles with fresh monolayer of Hep‐2 cells.…”

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…Thus, Azizi et al [92] developed a new viral quantitative CE (viral qCE) ( Figure 10) to determine: 1) the amount of intact virus particles (IVP); 2) the number of DNA contamination; and 3) the level of viral degradation after sonicating, vortexing, and freeze-thaw cycles, completed in a few hours with a wide range of virus concentrations (10 8 -10 13 ivp/mL). For this technique, CZE-LIF is used to separate IVPs from DNA and RNA impurities, and sample DNA and RNA is stained by YOYO-1 dye.…”