Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Buenos Aires, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, 1425, Buenos Aires, Argentina.
Appl Microbiol Biotechnol. 2017 May;101(10):4289-4298. doi: 10.1007/s00253-017-8244-2. Epub 2017 Mar 29.
Enteric viruses are pathogens associated with food- and waterborne outbreaks. The recovery of viruses from food or water samples is affected by the procedures applied to detect and concentrate them. The incorporation of an internal process control virus to the analyses allows monitoring the performance of the methodology. The aim of this study was to produce a recombinant adenovirus (rAdV) and apply it together with bacteriophage PP7 as process controls. The rAdV carries a DNA construction in its genome to differentiate it from wild-type adenovirus by qPCR. The stability of both control viruses was evaluated at different pH conditions. The rAdV was stable at pH 3, 7, and 10 for 18 h. PP7 infectious particles were stable at pH 7 and showed a 2.14 log reduction at pH 10 and total decay at pH 3 after 18 h. Three virus concentration methods were evaluated: hollow-fiber tap water ultrafiltration, wastewater ultracentrifugation, and elution-PEG precipitation from lettuce. Total and infectious viruses were quantified and their recoveries were calculated. Virus recovery for rAdV and PP7 by ultrafiltration showed a wide range (2.10-84.42 and 13.54-84.62%, respectively), whereas that by ultracentrifugation was 5.05-13.71 and 6.98-13.27%, respectively. The performance of ultracentrifugation to concentrate norovirus and enteroviruses present in sewage was not significantly different to the recovery of control viruses. For detection of viruses from lettuce, genomic copies of PP7 were significantly more highly recovered than adenovirus (14.74-18.82 and 0.00-3.44%, respectively). The recovery of infectious virus particles was significantly affected during sewage ultracentrifugation and concentration from lettuce. The simultaneous use of virus controls with dissimilar characteristics and behaviors might resemble different enteric viruses.
肠病毒是与食源性和水源性暴发相关的病原体。从食物或水样中回收病毒的效果受到用于检测和浓缩病毒的程序的影响。将内部过程控制病毒纳入分析中，可以监测方法的性能。本研究的目的是生产重组腺病毒(rAdV)，并将其与噬菌体 PP7 一起用作过程控制。rAdV 基因组中携带 DNA 构建体，通过 qPCR 将其与野生型腺病毒区分开来。在不同的 pH 条件下评估了两种对照病毒的稳定性。rAdV 在 pH 3、7 和 10 下稳定 18 小时。PP7 感染性颗粒在 pH 7 下稳定，在 pH 10 下降低 2.14 个对数，在 pH 3 下 18 小时后完全衰减。评估了三种病毒浓缩方法：中空纤维自来水超滤、废水超速离心和生菜洗脱-PEG 沉淀。定量测定总病毒和感染性病毒，并计算其回收率。超滤法回收 rAdV 和 PP7 的病毒回收率范围很宽(分别为 2.10-84.42%和 13.54-84.62%)，而超速离心法的回收率分别为 5.05-13.71%和 6.98-13.27%。与从污水中浓缩诺如病毒和肠道病毒的回收相比，超速离心浓缩污水中存在的诺如病毒和肠道病毒的性能没有显著差异。对于从生菜中检测病毒，PP7 的基因组拷贝数明显高于腺病毒(分别为 14.74-18.82%和 0.00-3.44%)。污水超速离心浓缩和从生菜中浓缩时，感染性病毒颗粒的回收率受到显著影响。同时使用具有不同特征和行为的病毒对照物可能类似于不同的肠道病毒。
