Degradation of Poliovirus Sabin 2 Genome After Electron Beam Irradiation

Abstract

Objectives: Most antiviral vaccines are created by inactivating the virus using chemical methods. The inactivation and production of viral vaccine preparations after the irradiation of viruses with accelerated electrons has a number of significant advantages. Determining the integrity of the genome of the resulting viral particles is necessary to assess the quality and degree of inactivation after irradiation.

Methods: This work was performed on the Sabin 2 model polio virus. To determine the most sensitive and most radiation-resistant part, the polio virus genome was divided into 20 segments. After irradiation at temperatures of 25 °C, 2-8 °C, -20 °C, or -70 °C, the amplification intensity of these segments was measured in real time.

Results: The best correlation between the amplification cycle and the irradiation dose at all temperatures was observed for segment 3D, left. Consequently, this section of the poliovirus genome is the least resistant to the action of accelerated electrons and is the most representative for determining genome integrity. The worst dependence was observed for the VP1 right section, which, therefore, cannot be used to determine genome integrity during inactivation. The electrochemical approach was also employed for a comparative assessment of viral RNA integrity before and after irradiation. An increase in the irradiation dose was accompanied by an increase in signals indicating the electrooxidation of RNA heterocyclic bases. The increase in peak current intensity of viral RNA electrochemical signals confirmed the breaking of viral RNA strands during irradiation. The shorter the RNA fragments, the greater the peak current intensities. In turn, this made the heterocyclic bases more accessible to electrooxidation on the electrode.

Conclusions: These results are necessary for characterizing the integrity of the viral genome for the purpose of creating of antiviral vaccines.

Keywords: accelerated electrons; biosensor; electrochemical analysis; genome degradation; poliomyelitis virus.

Figure 1

Diagram of the location of PCR segments on the genome of the Sabin strain type 2 of poliovirus. The diagram shows the position of the 5′-end of each of the regions encoding viral proteins. UTR is the non-translating region.

Figure 2

Representative photos of light microscopy show a monolayer of Vero cells during the control of residual infectivity. Inactivated sample, example of Vero cell monolayer after incubation with an inactivated sample of poliovirus strain Sabin type 2 (irradiated with accelerated electrons at a temperature of 2–8 °C and a dose of 25 kGy). Negative control, an intact monolayer of Vero cells that did not undergo treatment with the studied samples or virus. Viral control, the state of the monolayer of Vero cells after infection with poliovirus strain Sabin type 2 with 10 TCID₅₀/mL infectious titer. Scale bars in the images correspond to 250 µm.

Figure 3

RNA quality assessment in treated poliovirus. Total RNA was isolated from (A) irradiated samples or (B) samples inactivated by formaldehyde or beta-propiolactone. RNA integrity and quality (IQ) was measured by fluorescence and scored in arbitrary units (AU). N = 3. Agarose gel electrophoresis showing the degradation of viral RNA (C) after irradiation and (D) after chemical inactivation.

Figure 4

The dependence of the cycle threshold (Ct) on the irradiation dose. (A) The R² value showing the sensitivity of poliovirus RNA irradiated at different temperatures to accelerated electrons. The Sabin 2 strain samples were irradiated with accelerated electrons within the range of 10–25 kGy (50 mEV). Total RNA was isolated and subjected to qPCR. Plots of Ct versus irradiation dose were made and R² values were calculated. Black horizontal dashed line shows R² = 0.7. Plots of Ct versus irradiation dose for (B) the most representative 3D left and (C) the least representative VP1 right sites of viral RNA. The plots for the other viral RNA segments are presented in Figure S1 in the Supplementary File.

Figure 5

Cycles threshold (Ct) for Poliovirus genome segments after inactivation with formaldehyde or beta-propiolactone.

Figure 6

The dependence of the I/C signals for poliovirus Sabin 2 RNA on the irradiation doses at different temperatures to accelerated electrons: (A) 2–8 °C; (B) 25 °C; (C) −20 °C; (D) −70 °C. Plot of the I/C poliovirus irradiation doses with the best R² = 0.93 for the temperature to accelerated electrons of 2–8 °C. The zero point reflects the DPV signal of intact (control) poliovirus RNA without irradiation procedure. I—peak current, µA, C—RNA concentration, µg/mL.