Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method

Authors: Tian, Peng; WANG, DAPENG - Shanghai Jiaotong University; Yang, David

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 11/26/2013
Publication Date: 2/1/2014
Citation: Tian, P., Wang, D., Yang, D. 2014. Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method. Meeting Abstract. 172:76-82.

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. 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. Prior de-activation assays using Tulane Virus (TV), a culture-able analogue of HuNoV, have shown that there is little-to-no-correlation between the virus counts obtained from TCID50 assays versus the virus signal obtained from the qRT-PCR assay. While qRT-PCR is effective at quantitating the integrity of the targeted amplicon, it cannot detect critical damage to other areas of the viral genome outside of the qRT-PCR target amplicon, and most notably, it cannot detect critical degradation of the capsid required for binding and infection. Therefore qRT-PCR is only capable of detecting a subset of de-activating injuries/degradations to the virus, and its quantitation is inflated by false positives from otherwise de-activated/degraded virions. A new technique pioneered by Dancho, et al. involves isolating viruses using their functional receptors, followed by qRT-PCR. Virus samples are incubated in a container prior-prepared with immobilized receptor targets. Viruses become immobilized to the container by receptor binding, in-effect screening for functional receptors while isolating and concentrating the viral particles from the sample medium. The sample medium is then removed, and the viral RNA is released/extracted from the immobilized viruses into the container. The released viral RNA can be quantitated for the target amplicon by qRT-PCR, which in some cases can also be done in the very same container the virus was initially immobilized to. While this technique cannot eliminate the false positives arising from critical damage to other areas of the viral genome outside of the qRT-PCR target amplicon, it does address the loss-of-infectivity that arises from critical degradation of the capsid. We repeated the prior TV de-activation assays using this technique, and found improved consistency between the receptor-capture qRT-PCR assays and that of TCID50 assays. Using HBGAs as the immobilized binding receptor, we applied the receptor-capture qRT-PCR technique (HBGA-cqRT-PCR) to de-activation assays of HuNoV, and arrived at an improved map of HuNoV’s de-activation response.

Technical Abstract: Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Due to the inability to cultivate HuNoVs, it has been a challenge to determine their infectivity. Quantitative real-time RT-PCR (qRT-PCR) is widely used in detecting HuNoVs. However, qRT-PCR only detects the presence of viral RNA and does not indicate viral infectivity. A virus could lose its infectivity by damage to its viral capsid, but the viral RNA may still persist to be detected by qRT-PCR. Likewise, even if a treatment can lethally-damage the viral RNA, that damage may occur at one-or-more regions outside of the short span of the targeted qRT-PCR amplicon, so that too may still persist to be detected by qRT-PCR. For the purpose of detecting viable and infectious HuNoVs, conventional qRT-PCR is hindered by these false positives. Human blood group antigens (HBGAs) have been identified previously as receptors for human and some animal NoVs. Our method employs HBGAs as a container-affixed capture agent that serves as both the means to concentrate virus from solution, as well as that to select intact capsids. Previously, we have demonstrated that the inactivation of Tulane virus measured by the HBGA based capture qRT-PCR (HBGA-cqRT-PCR) were consistent with results measured by TCID50. In this paper, we demonstrated that HBGA-cqRT-PCR method could effectively concentrate HuNoV and distinguish the inactivated virus from infectious virus. HuNOV could be fully inactivated by heat at 72oC for 4 minutes, chlorine at a final concentration of 16 ppm for 10 minutes, and ethanol at a final concentration of 70% for 20 seconds.