X-ray inactivation of RNA viruses without loss of biological characteristics

Abstract

In the event of an unpredictable viral outbreak requiring high/maximum biosafety containment facilities (i.e. BSL3 and BSL4), X-ray irradiation has the potential to relieve pressures on conventional diagnostic bottlenecks and expediate work at lower containment. Guided by Monte Carlo modelling and in vitro 1-log₁₀ decimal-reduction value (D-value) predictions, the X-ray photon energies required for the effective inactivation of zoonotic viruses belonging to the medically important families of Flaviviridae, Nairoviridae, Phenuiviridae and Togaviridae are demonstrated. Specifically, it is shown that an optimized irradiation approach is attractive for use in a multitude of downstream detection and functional assays, as it preserves key biochemical and immunological properties. This study provides evidence that X-ray irradiation can support emergency preparedness, outbreak response and front-line diagnostics in a safe, reproducible and scalable manner pertinent to operations that are otherwise restricted to higher containment BSL3 or BSL4 laboratories.

Figure 1

Effect of X-ray beam filtration on ZIKV inactivation. Monte Carlo simulation of beam filtration effect on (a) X-ray photon energy spectra showing beam hardening^, and (b) dose-to-depth penetration of photons in water at 220 keV, 18.2 mA using MCNP6.1 model in (c) the irradiation chamber showing the sealed virus specimens with packaging dimensions of 10 cm × 8 cm × 1 cm (L x W x D) at a distance of 22.6 cm within a 37.5° irradiation cone produced by the MXR-225/26 tube. Incremental X-ray irradiation of ZIKV at the above conditions showing (d) the effect of beam filtration on ZIKV RNA genomic-equivalent copies using RT-qPCR and (e) Infectivity titer of ZIKV by TCID₅₀ assay. All data was acquired by accounting for a sample-to-source distance of 22.6 cm and each data point represents the mean of triplicate independent runs.

Figure 2

Inactivation of viral pathogens using X-rays. (a) X-ray D-values showing the dose required to inactivate 1-log₁₀ of virus at 220 keV, 17.5 mA with 0.2 mm Al filtration. In vitro CPE was not observed in an infection-sensitive cell substrate after treatment with 1 mL of virus and following 3 passages at predicted inactivation doses. X-ray D-values were then compared to gamma inactivation data produced under similar sample conditions, for members of the same genus. (b) ZIKV infectivity in A129 mice after intravenous administration of irradiated ZIKV and (c) RVFV infectivity in BALB/c mice after intramuscular administration of irradiated RVFV; data shows 100% survival of mice for receiving viruses that had been irradiated with inactivating doses of X-rays at predicted D-values, in agreement with in vitro passage data in the appropriate cell line model.

Figure 3

Nucleic acid and protein analysis of X-ray inactivated virus. (a) Box plot of N50 contig assembly statistics for RVFV and ZIKV, showing no statistical differences (Mann–Whitney U, two-sided) between viral contig re-assembly across radiation doses (0 to 24 kGy), (b) NGS mapping data demonstrating similar depth of coverage when comparing sequencing of non-irradiated and irradiated ZIKV-X and RVFV-X and (c) regression analysis of variants induced through X-ray irradiation across dose increments, showing non-significant correlations (r ≤ 0.32 ). The effect of irradiation on viral proteins by (d) Western blot detection of ZIKV envelope illustrating uniform sensitivity of detection, (e) abrogation of competition ELISA signal of RVFV anti-NP using X-ray irradiated antigen (n = 3 per dose). (f) two-fold serial dilutions of whole-virus RVFV irradiated with 18.2 kGy of X-rays inhibits the binding of anti-NP in commercially sourced competition ELISA.