Purpose: This study evaluates the impact of virus inoculum matrix and titer when enzymatic pre-treatment is employed prior to RT-PCR to predict HuNoV infectivity after heat inactivation. Plaque assay and negative staining electron microscopy (EM) of thermally inactivated murine norovirus (MNV-1) is also included for comparison.
Methods: MNV-1 (5.5-6.4 log PFU/ml) in 12 x 32 mm vials was subjected to 50, 56 and 60°C temperatures for time points between 30 sec to 2 hr. Virus inactivation was visualized by EM and D-values were determined after plaque assay. Low and high titer (approx. 1.5 and 5.5 log PFU/ml, respectively) untreated or heat-inactivated (99oC for 5 mins) virus stocks were subjected to enzymatic pre-treatment. The impact of inoculum matrix, was also evaluated using stocks that were sucrose purified, ultrafiltered, and syringe-filtered (SP/UF/SF), just (SP/UF), or SP/UF with the addition of 5% fetal bovine serum (FBS). Ct values were determined by RT-qPCR.
Results: We report D-values of 77.5, 11.3 and 2.1 min for 50, 56, and 60°C, respectively. For thermally inactivated high titer virus stocks, Average Ct value RDs between the enzyme-treated and untreated samples were 10.18 ± 6.03, 9.73 ±7.41 and 2.31 ±2.04 , for SP/UF/SF, SP/UF, and SP/UF+5% FBS stocks, respectively. EM results revealed intact viral particles in untreated samples, but no discernible intact particles after thermal inactivation.
Significance: This study suggests that both the virus suspension matrix and the virus titer impact RT-PCR results following enzyme pretreatment in thermal inactivation studies. Therefore, these factors should be considered before applying this method in HuNoV detection studies.