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

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 (TCID). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID reductions of 0.3, 4.4 and 5.9 log were observed. UV exposure (40/100/1000 mJ/cm) resulted in 1.1, 2.5 and 5.9 log reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID. On average, RT-qPCR with pre-treatments deviated by 1.1-1.3 log from TCID. For UV light, long-range PCR was closest to TCID results. Long-range reductions deviated from TCID by ≤0.1 log 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 log from TCID. After chlorination the molecular methods repeatedly deviated from TCID by >1.0 log, Overall, each method needs to be further optimized for the individual types of inactivation treatment.