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To detect less prevalent viruses, such as wild-type polioviruses in sewage from a highly immunized community, a method was developed to efficiently recover viruses and remove PCR inhibitors. The method consisted of initial separation of solids from liquid, followed by solvent extractions, polyethylene glycol precipitations, Sephadex G-200 chromatography, and guanidinium isothiocyanate (GIT) extraction. To elute viruses from the separated solids, 0.5 M threonine (pH 7.5) was as efficient as 3% beef extract but conferred no PCR inhibition. In samples that were concentrated approximately 1,000-fold, 21% of the initially seeded viruses were recovered. When poliovirus type 3 (PV3) Sabin strain at low levels and PV1 LSc strain at high levels were seeded in raw sewage, PV3 was specifically detected in the final sample concentrates at sensitivities of 14 PFU by direct PCR and 0.7 PFU by GIT extraction-PCR. While applying the method to international airplane sewage, which contains high levels of solids as well as commercial sanitizers, 44% (7 of 16) of the samples were found to harbor enteroviruses by both cell culture infectivity and pan-enterovirus PCR analyses. Nucleotide sequencing of the PCR products revealed that multiple enterovirus genotypes were amplified from each final sewage concentrate, whereas the fewer virus genotypes detected by cell culture infectivity were probably the better growing strains. By this method, we demonstrated that air travel may contribute to the intercontinental dissemination of enteric pathogens.

Typical environmental sample concentration procedures developed to purify virions are not always compatible with reverse transcription-polymerase chain reaction (RT-PCR). The processing steps of polyethylene glycol (PEG) precipitation and ultrafiltration were found to enrich not only virions but also certain RT-PCR inhibitors. Inhibitors were eliminated by a single-step guanidinium isothiocyanate (GIT) extraction to purify and precipitate viral genomic RNAs immediately prior to RT-PCR. The detection sensitivity of GIT extraction--RT-PCR was found to be 0.6-0.003 50% tissue culture infectious doses (TCID50) for 9 enteroviruses in infected cell extracts. When sewage, concentrated up to 385,000-fold and seeded with 1-3 plaque-forming units of poliovirus, was extracted with GIT solution, viruses were detectable in samples originally judged negative by direct RT-PCR without GIT extraction. Eleven waste samples (3 sewage, 5 latrine solids, 2 gauze pads extracts and 1 stool) processed by a series of steps that included PEG precipitation, solvent extractions, and Sephadex G-200 chromatography were examined for enteroviruses by RT-PCR, both with and without GIT extraction. GIT extraction eliminated sample inhibitory substances and increased the proportion of enterovirus positive samples from 3 to 7 of 11. GIT extraction in conjunction with virion concentration improves RT-PCR detection of viruses in sewage and other fecal wastes.

Sensitive and specific methods are needed to detect hepatitis A virus (HAV) and other human enteroviruses in environmental samples such as drinking water and foods. Clones of cDNA encoding the 5'-most 1 kb of the HAV and coxsackievirus B3 (CB3) genomes were subcloned into T7/SP6 RNA transcription vectors. In vitro transcribed RNA from the T7 promoter detected their respective HAV or CB3 genomic RNA. Conversely, SP6 transcripts detected viral negative-stranded RNA but not the genome. When both ssRNA probes were tested at high temperature (65 degrees C), they did not hybridize with intracellular RNAs from 6 primate cell cultures used for isolation of HAV and other enteroviruses. The HAV probe did not hybridize with 13 different enteroviruses but detected as little as 500-1000 infectious units of the 7 strains of HAV tested. Conversely, the CB3 probe showed strong homology with all 13 enteroviruses tested but not HAV. The probes were used to detect HAV and other enteroviruses in water samples after virus amplification in cell culture. HAV was detected in water samples obtained during a waterborne hepatitis outbreak using the ssRNA probe. These samples were negative for HAV by direct solid phase radioimmunoassay and were not positive by immunoassays of inoculated cell cultures until several weeks of propagation. The CB3 ssRNA probe detected enteroviruses in samples of surface water and drinking water that were negative for cytopathic effects in inoculated cell cultures.