Effect of handling and storage conditions and stabilizing agent on the recovery of viral RNA from oral fluid of pigs

There is an increasing interest in using oral fluid to determine herd health and documenting the circulation of viruses in commercial swine populations but little is known about the stability of viruses in oral fluid. Hepatitis E virus (HEV) is a zoonotic virus which is widespread in swine herds. Information on optimal handling methods such as heat treatments, freezing and RNA stabilization agents is needed to prevent or minimize degradation of viral RNA by degradative enzymes. The objectives of the study were to determine optimum handling conditions of the oral fluid before RNA extraction and to compare the performance of the RNeasy Protect Saliva Mini kit, which contains a stabilizing agent, with that of the QIAamp Viral RNA Mini kit, which does not contain a stabilizing agent. Preliminary studies with oral fluid inoculated with HEV indicated that a heat treatment of 60°C for 15min was detrimental to HEV RNA. HEV was recovered from 25/25 and 24/25 samples of oral fluid when samples were incubated for ≤24h at 4°C and 30days at -20°C, respectively, without a stabilizing agent and extracted with the QiaAMP kit. In contrast, HEV RNA was detected in 16/25 and 11/25 samples when samples were incubated with a stabilizing agent for 24h at 37°C and 30days at -20°C, respectively, and extracted with the RNeasy Protect Saliva kit. Moreover, the mean number of genome copies/ml of HEV recovered from oral fluid stored at -20°C without the stabilizing agent was 2.9 log units higher than oral fluid stored at -20°C in the presence of the stabilizing agent. The recovery of RNA from HEV, F-RNA coliphage MS2 and murine norovirus (MNV), which are surrogates for norovirus, was significantly greater when oral fluid was incubated for 24h at 4°C than when oral fluid was stabilized with RNAprotect Saliva Reagent for 24h at 37°C, where the relative differences between the two processes were 1.4, 1.8, and 2.7 log genome copies/ml for MS2, MNV, and HEV, respectively. The findings suggest that it is unnecessary to stabilize oral fluid from swine for the detection of viral RNA, provided the samples are stored at 4°C or frozen at -20°C, and that the RNeasy Protect Saliva Mini kit did not perform well for the detection of viral RNA.