Membrane adsorption with direct cell culture combined with reverse transcription-PCR as a fast method for identifying enteroviruses from sewage

Abstract

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 Alpha9 (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.

FIG. 1.

(a) Representative results for UG₅₂-Uc₅₃ (435-bp) amplicons and 292-222 (350-bp) amplicons for isolates A1, A2, A3, and A4. Lane M contained a molecular weight marker (φX174 HaeIII-generated restriction fragments; Gibco BRL). (b) Representative results of RFLP analysis of UC₅₃-UG₅₂-produced RT-PCR amplicons of the enterovirus isolates with the HpaII restriction endonuclease (for a description, see the text). All of the isolates except one (isolate A5 [data not shown]) belonged to the same genetic subcluster, and the restriction profile comprised four HpaII-generated fragments with lengths of 213, 149, 55, and 18 nucleotides. The lane on the left contained a molecular weight marker (φX174 HaeIII-generated restriction fragments; Gibco BRL).

FIG. 2.

Dendrogram showing the relationships among enterovirus environmental strains isolated in the present study and reference and other clinical strains, as determined by using the partially sequenced VP1-encoding region.