Differences in Viral Disinfection Mechanisms as Revealed by Quantitative Transfection of Echovirus 11 Genomes

Abstract

Virus inactivation mechanisms can be elucidated by methods that measure the loss of specific virus functionality (e.g., host attachment, genome internalization, and genome replication). Genome functionality is frequently assessed by PCR-based methods, which are indirect and potentially inaccurate; genome damage that affects detection by high-fidelity PCR enzymes may not adversely affect the ability of actual cellular enzymes to produce functional virus. Therefore, we developed here a transfection-based assay to quantitatively determine viral genome functionality by inserting viral RNA into host cells directly to measure their ability to produce new functional viruses from damaged viral genomes. Echovirus 11 was treated with ozone, free chlorine (FC), UV light at 254 nm (UV₂₅₄), or heat, and then the reductions in genome functionality and infectivity were compared. Ozone reduced genome functionality proportionally to infectivity, indicating that genome damage is the main mechanism of virus inactivation. In contrast, FC caused little or no loss of genome functionality compared to infectivity, indicating a larger role for protein damage. For UV₂₅₄, genome functionality loss accounted for approximately 60% of virus inactivation, with the remainder presumably due to protein damage. Heat treatment resulted in no reduction in genome functionality, in agreement with the understanding that heat inactivation results from capsid damage. Our results indicate that there is a fundamental difference between genome integrity reductions measured by PCR enzymes in previous studies and actual genome functionality (whether the genome can produce virus) after disinfection. Compared to PCR, quantitative transfection assays provide a more realistic picture of actual viral genome functionality and overall inactivation mechanisms during disinfection.IMPORTANCE This study provides a new tool for assessing virus inactivation mechanisms by directly measuring a viral genome's ability to produce new viruses after disinfection. In addition, we identify a potential pitfall of PCR for determining virus genome damage, which does not reflect whether a genome is truly functional. The results presented here using quantitative transfection corroborate previously suggested virus inactivation mechanisms for some virus inactivation methods (heat) while bringing additional insights for others (ozone, FC, and UV₂₅₄). The developed transfection method provides a more mechanistic approach for the assessment of actual virus inactivation by common water disinfectants.

Keywords: UV254; disinfection; echovirus 11; enterovirus; free chlorine; genome functionality; genome integrity; heat inactivation; ozone; transfection.

FIG 1

Transfection of RNA extracted from different concentrations of infectious viruses (IV) for all samples (a) and for the three samples used to test repeatability (b). In panel a, the dashed line represents a 1:1 relationship, while the dotted line represents the linear regression calculated between IV per ml and transfectable genome units (TGUs) per ml. The distance between the two lines indicates the efficiency of the transfection assay. The slope of the dotted line indicates the proportionality between IV and TGU. The “×” symbol represents a sample that was quantifiable for infectious virus but not quantifiable by the transfection method. For the graph in panel b, the x axis represents categorical values of log₁₀ IV per ml; points are separated in the x direction for a given log₁₀ IV value to display overlapping points only. Error bars represent 95% CI for TGU values determined by the MPN method.

FIG 2

Comparison of log₁₀ reductions of infectious E11 (IV) and log₁₀ reductions of genome functionality measured in transfectable genome units (TGUs) after inactivation by ozone (a), free chlorine (b), UV₂₅₄ (c), and heat (d). C₀ and C indicate the concentrations of TGU or IV prior to and after inactivation, respectively. Dashed lines represent a 1:1 relationship. The dotted line represents the linear regression calculated between infectious virus reductions and TGU reductions. For the graph panel b, FC concentrations are shown separately for 2 mg/liter (open circles), 2.5 mg/liter (light circles), and 3 mg/liter (dark circles); the regression line was calculated for all FC concentrations together.