The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity

Abstract

Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID₅₀). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID₅₀ reductions of 0.3, 4.4 and 5.9 log₁₀ were observed. UV exposure (40/100/1000 mJ/cm²) resulted in 1.1, 2.5 and 5.9 log₁₀ reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log₁₀. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID₅₀. On average, RT-qPCR with pre-treatments deviated by 1.1–1.3 log₁₀ from TCID₅₀. For UV light, long-range PCR was closest to TCID₅₀ results. Long-range reductions deviated from TCID₅₀ by ≤0.1 log₁₀ for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log₁₀ from TCID₅₀. After chlorination the molecular methods repeatedly deviated from TCID₅₀ by >1.0 log₁₀, Overall, each method needs to be further optimized for the individual types of inactivation treatment.