Infectious Virus Identification Assay And Its Application Based On Murine Norovirus

Human noroviruses(Hu No Vs)are one of the main pathogens that cause global outbreaks of non-bacterial gastroenteritis,which can be spread through the fecal-oral route.So far,Hu No Vs still lack a stable and reliable in-vitro culture system,and its confirmation relies on molecular biological testing methods.Real-time quantitative reverse transcription-polymerase chain reaction(RT-q PCR),as the international gold standard for Hu No Vs detection,can simultaneously amplify free RNA,partially damaged RNA and RNA inside damaged capsids.Thus,RT-q PCR assay cannot effectively distinguish infectious and non-infectious viruses,potentially overestimating amounts of infectious viruses.Establishing an infectious virus identification method based on Hu No Vs surrogates and further applying it to identify infectious Hu No Vs has become a popular research trend now.Thus,this research selected murine norovirus(MNV)that can be cultured in vitro as the subject,aiming to establish a method that can be applied to identify the infectious virus in wakame,and to provide a theoretical basis for the future application in Hu No Vs.The main outcomes of this study are as follows:1.The damage of MNV heated at 55?95?was observed by transmission electron microscope.To obtain the normalized amplicon decay rate constant(k_i,norm),the whole genome of MNV was divided into 31 of amplification regions,before a mathematical model was established between the heating temperature and the reduction of gene titer of each amplicon.Thereafter,the thermal damage of whole-genome was analyzed and the heat-sensitive nucleotide region was determined.Finally,long-range RT-q PCR assay targeting the most heat-sensitive nucleotide region was used to monitor the thermal inactivation of MNV at different titers under 55?95?.The results showed that the capsid protein of MNV was denatured or less damaged after heating at 55?75°C.But the higher temperature(85?95?)would further damage the capsid and even degrade the protein into small subunits.The nucleic acid damage induced by heat treatment was unevenly distributed throughout the genome of MNV.The nucleotide regions of 5'-UTR,ORF1 head(259?1147)and tail(2873?4586)were the main damaged area,and the region of 4289?4586 was most sensitive to temperature(k_i,norm=3.16×10^-4 K^-1 base^-1).Long-range RT-q PCR targeting this region(4289?4586)was unable to completely reduce the gene titer of MNV killed by heat-treatment at 55?95°C,but its ability to exclude amplification signals of heat-killed MNV was stronger than RT-q PCR assay.2.To further remove the amplification signals derived from heat-inactivated MNV,viability markers were introduced into the system to establish PMAxx/Pt Cl₄-long-range RT-q PCR assays.And then,the q PCR amplicons and incubation conditions of viability markers were optimized.Using TCID₅₀ assay as a control,the optimized PMAxx/Pt Cl₄-long-range RT-q PCR assays were used to monitor the heat-inactivation of MNV at 56,75 and 85°C.The results showed that incubating heat-killed MNV with 50?M PMAxx at 25°C in the dark for 15 min and then photoactivating for 10 min was the best condition for PMAxx using.Incubating 2500?M Pt Cl₄ with heat-killed MNV for 30min at 4°C was the best condition for Pt Cl₄ using.The longer q PCR amplicons improved the accuracy of PMAxx/Pt Cl₄-long-range RT-q PCR in infectious MNV identification,and 425 bp was the best target amplicon to eliminate false-positive results from non-infectious MNV.PMAxx/Pt Cl₄-long-range RT-q PCR assays were more suitable for enumerating the numbers of infectious viruses in viral suspension heated at high temperature(10?15 min),but this method cannot effectively evaluate the heat-inactivation of MNV at 56?.3.In order to improve the efficiency of PMAxx/Pt Cl₄-long-range RT-q PCR assays in wakame matrices,PMA Enhancer,Triton X-100 and Tween-20 were tested in wakame concentrate.And then,the further optimized method was used to evaluate the heat-inactivation of MNV(75?95?)inoculated on the surface of wakame.The results showed that PMA Enhancer was the best reagent to eliminate the matrix interferences and to enhance the effectiveness of viability markers in identifying infectious MNV.The limit of quantification of PMAxx/Pt Cl₄-PMA Enhancer-long-range RT-q PCR assays was 0.31 log TCID₅₀/5 g in the wakame matrix.In this matrix,PMAxx-PMA Enhancer-long-range RT-q PCR assay was better than Pt Cl₄-PMA Enhancer-long-range RT-q PCR assay in identifying infectious MNV,and the previous one eliminated the amplification signals of non-infectious MNV(killed by 75?95?heat-treatment for 5min)at a lower titer(3?4 log TCID₅₀).When MNV was inoculated in wakame with a higher titer(4?5 log TCID₅₀),PMAxx-PMA Enhancer-long-range RT-q PCR assay effectively monitored the heat-inactivation of MNV heated at 85?95?for 15 min.But this approach was unable to completely eliminate gene titers of non-infectious MNV heated at 75°C for 15 min.In conclusion,PMAxx-PMA Enhancer-long-range RT-q PCR holds great potential for its application in complex food matrices(such as wakame).It also can be used as a simple and highly sensitive method to quantify infectious viruses in food safety risk analysis and assessment.