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Applied and Environmental Microbiology, January 2005, p. 72-79, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.72-79.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Membrane Adsorption with Direct Cell Culture Combined with Reverse Transcription-PCR as a Fast Method for Identifying Enteroviruses from Sewage

Department of Virology, National Reference Enteroviruses Center, Hellenic Pasteur Institute,¹ Technological Educational Institution, Athens,² Department of Microbiology, Medical School, University of Ioannina, Ioannina,³ Microbiology-Virology Laboratory, Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece,⁴ Microbiology Section, State General Laboratory, Ministry of Health, Nicosia, Cyprus⁵

Received 17 June 2004/ Accepted 18 July 2004

We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5' untranslated region (5'-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie A9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5'-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates.