Muscillo M, La Rosa G, Marianelli C, Zaniratti S, Capobianchi M R, Cantiani L, Carducci A
Laboratorio di Igiene Ambientale, Department of Environmental Hygiene, Istituto Superiore di Sanita', Viale Regina Elena 299, Rome, Italy.
Water Res. 2001 Feb;35(2):548-56. doi: 10.1016/s0043-1354(00)00282-7.
The frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. In order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (RT-PCR) was set up to detect reoviruses. Two primers were engineered to amplify a 538 base pair fragment of the Sigma 2 gene. Reovirus strains obtained from ATCC (Jones, Lang, Dearing, Abney, NC-TEV, SV59 and SV12) were used as references. Twenty-four samples of 101 were collected from two beaches of the Adriatic sea and 12 from the neighbourhood of Fano Harbour Channel. The presence of environmental reoviruses was tested on both concentrated seawater samples and lysates of BGM cells infected with the concentrated seawater samples. The new method was used in parallel with the detection of a 3:3:4 electrophoretic pattern of reovirus RNA in polyacrylamide gel electrophoresis (PAGE). Enterovirus and bacteria were also screened in compliance with EEC directives. No enteroviruses were isolated, and it was not attributable to reovirus interference. All the reovirus found by PAGE (8/72) were confirmed by RT-PCR, while several genomes (14/72) were detected only by RT-PCR. Presumptive methods of virus identification, that is CPE on BGM cells and haemagglutination test, were not able to detect them. The specificity of RT-PCR products was checked by direct nucleotide sequence analyses of the amplicons. The phylogenetic analyses showed heterogeneous taxa including human and animal reoviruses, with strong evidence that they were spreading consistently from the Harbour-Channel. This novel approach for reovirus detection will be very useful as a trace route of faecal pollution; more importantly, it could be very useful in contributing to the creation of a databank of circulating enteric viruses.
呼肠孤病毒在环境样本中的频繁出现可能是干扰肠道病毒检测的一个潜在来源，尤其是在需要通过细胞培养分离肠道病毒时。为了评估基于新病毒的休闲用水水质标准执行标准，建立了一种基于广泛逆转录聚合酶链反应（RT-PCR）的新方法来检测呼肠孤病毒。设计了两种引物来扩增西格玛2基因的一个538碱基对片段。从美国典型培养物保藏中心获得的呼肠孤病毒株（琼斯株、朗株、迪林株、阿伯尼株、NC-TEV株、SV59株和SV12株）用作参考。从亚得里亚海的两个海滩采集了101份样本中的24份，从法诺港航道附近采集了12份。对浓缩海水样本以及感染了浓缩海水样本的BGM细胞裂解物进行了环境呼肠孤病毒的检测。该新方法与在聚丙烯酰胺凝胶电泳（PAGE）中检测呼肠孤病毒RNA的3:3:4电泳图谱同时使用。还按照欧盟指令对肠道病毒和细菌进行了筛查。未分离出肠道病毒，这并非呼肠孤病毒干扰所致。通过PAGE发现的所有呼肠孤病毒（8/72）均经RT-PCR确认，而一些基因组（14/72）仅通过RT-PCR检测到。病毒鉴定的推定方法，即在BGM细胞上的细胞病变效应和血凝试验，无法检测到它们。通过对扩增子进行直接核苷酸序列分析来检查RT-PCR产物的特异性。系统发育分析显示存在包括人类和动物呼肠孤病毒在内的异质类群，有力证据表明它们一直在从港道持续传播。这种检测呼肠孤病毒的新方法作为粪便污染的追踪途径将非常有用；更重要的是，它可能对建立循环肠道病毒数据库非常有帮助。
