Garcia Lucas A T, Nascimento Mariana A, Barardi Célia R M
Laboratório de Virologia Aplicada, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, 88040-900, Brazil.
Food Environ Virol. 2015 Mar;7(1):67-75. doi: 10.1007/s12560-014-9177-x. Epub 2014 Dec 21.
Shellfish depuration is a process that aims to eliminate pathogens from mollusk tissues. Seawater disinfection during the depuration process is important and ultraviolet (UV) light treatment is the most used method worldwide. Viral models are usually employed as surrogates of fastidious viruses in viability studies. The aim of this study was to employ methods based on green fluorescent protein (GFP) fluorescence and plaque forming units to detect, respectively, recombinant adenovirus (rAdV-GFP) and murine norovirus (MNV) artificially seeded in environmental matrices. These assays were applied to assess the inactivation of rAdV-GFP and MNV in seawater in recirculation shellfish depuration tanks with and without UV light treatment. Kinetics of rAdV GFP-expression was previously measured by UV-spectrophotometer. Flow cytometry (FC), fluorescence microscopy (FM), and plaque assay were used to determine virus titer and detection limits. The influence of the environmental matrix on the performance of the methods was prior determined using either drinking water or filtered seawater seeded with rAdV-GFP. Disinfection of seeded seawater was evaluated with and without UV treatment. The time of 24-h post-infection was established as ideal for fluorescence detection on rAdV-GFP infected cells. FC showed lower sensitivity, when compared to FM, which was similar to plaque assay. Seawater disinfection on depuration tanks was promising and rAdV-GFP declined 99.99 % after 24 and 48 h with and without UV treatment, respectively. MNV was completely inactivated after 24 h in both treatments. As conclusion, the depuration tanks were effective to inactivate rAdV-GFP and MNV and the UV disinfection treatment accelerated the process.
贝类净化是一个旨在从软体动物组织中消除病原体的过程。净化过程中的海水消毒很重要，紫外线（UV）光处理是全球最常用的方法。在生存能力研究中，病毒模型通常被用作难培养病毒的替代物。本研究的目的是采用基于绿色荧光蛋白（GFP）荧光和噬斑形成单位的方法，分别检测人工接种于环境基质中的重组腺病毒（rAdV-GFP）和鼠诺如病毒（MNV）。这些检测方法用于评估在有或无紫外线光处理的循环贝类净化池中，海水中rAdV-GFP和MNV的灭活情况。rAdV GFP表达的动力学先前已通过紫外分光光度计进行了测量。流式细胞术（FC）、荧光显微镜（FM）和噬斑测定法用于确定病毒滴度和检测限。预先使用接种了rAdV-GFP的饮用水或过滤海水来确定环境基质对这些方法性能的影响。评估了接种海水在有或无紫外线处理情况下的消毒情况。确定感染后24小时是检测rAdV-GFP感染细胞荧光的理想时间。与FM相比，FC显示出较低的灵敏度，而FM与噬斑测定法类似。净化池中的海水消毒前景良好，无论有无紫外线处理，rAdV-GFP在24小时和48小时后分别下降了99.99%。在两种处理中，MNV在24小时后均被完全灭活。总之，净化池对灭活rAdV-GFP和MNV有效，紫外线消毒处理加速了这一过程。
