Biology and Control of Human Pathogens on Fresh Produce
Location: Produce Safety and Microbiology Research
Title: Inactivation of Tulane virus, a novel surrogate for human norovirus
Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 1, 2012
Publication Date: N/A
Interpretive Summary: Human noroviruses (HuNoV) are pathogens of significant concern, but as of current they cannot be cultured, which limits the ways in which they can be studied. Surrogates for HuNoV have been used to this end, but they are not perfect and often differ from HuNoV in fundamental ways. Tulane virus (TV) is a recently-discovered candidate for being an experimental surrogate of HuNoV. The conditions required to inactivate TV is an important characteristic that can be suggestive of how well it can serve as a surrogate to HuNoV, but have not yet been thoroughly-documented. We have tested different types of inactivation conditions and agents across a range of intensities, and quantitated the results using the TCID50 assay. Molecular-based virus quantitation assays, notably qRT-PCR, have been used in lieu of in vivo/ex vivo assays for a number of reasons, including speed, ease, and feasibility. It is, however, questionable that what they quantitate is any accurate reflection of actual virus activity. Building on the results of our ex vivo assays, we tested the same inactivation conditions and agents to see if there is any correlation. We have found that TV is comparable to HuNoV and its existing surrogates in a variety of ways, and is closer to some than others for different methods of inactivation. We have also found that there is mostly little-to-no correlation between the virus counts obtained from the TCID50 assays and the virus signal obtained from the qRT-PCR assay, as the latter really only measures for the integrity of the targeted amplicon. This notably omits viral inactivation from degradation of the capsid required for binding and infection, not to mention critical damage to other areas of the viral genome not covered by the qRT-PCR target amplicon. We believe that a new technique that involves isolating viruses using their functional receptors, followed by qRT-PCR, is a molecular-based method that will better reflect the virus counts from the TCID50 assay.
Human noroviruses (HuNoVs) are the major cause of non-bacterial epidemics of gastroenteritis. Due to the inability to cultivate HuNoVs and the lack of an efficient small animal model, surrogates are used to study HuNoV biology. Two such surrogates, the feline calicivirus (FCV) and the murine norovirus (MNV), have been extensively studied but effort to seek for new surrogates is needed because neither of the two viruses are truly intestinal pathogens, although MNV could be detected in stools of mice. The newly described Tulane virus (TV) is a typical calicivirus (CV) that was isolated in stools of monkeys, cultivable in cell cultures, and recognizes human histo-blood group antigens (HBGAs), therefore, is a promising surrogate for HuNoVs. In this study, we evaluated the resistance or stability of TV under different physical and environment conditions by measuring a 50% reduction of tissue culture infective dose (TCID50) using the TV cell culture system. TV was fully inactivated (= three logs reduction in virus titer) after incubation at 63°C for 5 min or at 56°C for 30 min, or exposure to UV-C radiation at 60 mJ/cm2 or to 300 ppm of free chlorine for 10 min. TV is stable in pH 3 to 8 Significant reduction in virus titer was observed when pH changed to 2.5 or 9.0 and fully inactivated at pH10. TV is resistant to a low percent of EtOH (40% or lower) but was fully inactivated by 50-70% alcohol with a short exposure (20 sec). In contrast,a quantitative real time RT-PCR (qRT-PCR) was unable to detect or detect poorly a virus titer reduction between treated and untreated samples described in this study.