An immunomagnetic capture reverse transcription-PCR(IMC-RT-PCR) assay was evaluated to recover and detectenteric viruses in sewage and to remove PCR inhibitors. Theprocedure was applied along with a simple sample processingconsisting of an initial separation of solids followed by poly-ethylen glycol precipitation and solvent extraction. Thisprocedure reduced sample volumes by about 65-fold withouteliminating RT-PCR inhibitors. Paramagnetic beads coupledto pooled human immunoglobulins were used to simultan-eously capture poliovirus 1 (PV-1) and hepatitis A virus (HAV)from seeded sewage concentrates. The IMC was efficient inremoving PCR inhibitors and in further reducing samplevolumes by approximately 10-fold allowing the analysis of6–7 ml of sewage sample per RT-PCR reaction. The detectionlimits of IMC-RT-PCR from seeded concentrates were 0.1–1 PFU for PV-1 and 1 MPNCU for HAV. The describedprocedure could be applied successfully for the detection ofenteroviruses, HAV and rotaviruses in field sewage samples.

Key words: enteroviruses – hepatitis A virus – rotaviruses –immunomagnetic capture – PCR-sewage

Introduction

Enteric viruses including recognized viral pathogens areshed in human faeces and they have been detected insewage (Shieh et al. 1997; Pina et al. 1998; Gantzer et al. 1998). As viruses are not removed efficiently bysewage treatment, they can contaminate receiving

waters and cause water-borne diseases (Abad et al.1994; De Serres et al. 1999). In order to protect publichealth, simple and efficient methods are needed to carry out virological studies in water environments. The reverse transcriptase-polymerase chain reaction(RT-PCR) has been successfully applied for the detec-tion of RNA viruses in environmental samples (Shieh et al. 1997; Pina et al. 1998; Green and Lewis 1999).The RT-PCR is a very useful tool for the detection ofenteric viruses, including nonculturable viruses, due toits high specificity and sensitivity as well as its rapidprocessing time and low cost. The application of RT-PCR for enteric viruses detection in most environ-mental samples requires the concentration and purifica-tion of the sample because of the low virus concen-tration and the presence of amplification reactionnatural inhibitors. Different methods of virus extraction,concentration and purification have been developed toovercome these problems and to achieve the RT-PCRamplification of viruses in complex environmentalsamples such as sewage or sludge (Puig et al. 1994; Tsai et al. 1994; Shieh et al. 1997). However, most pro-cedures require extensive processing steps that increaseviral losses during manipulation and reduce detectionsensitivity.

Immunomagnetic capture (IMC) of viruses fromclinical and environmental samples has been describedas a method to increase PCR sensitivity (Monceyronand Grinde 1994; Jothikumar et al. 1998; Suñén andSobsey 1999; Bidawid et al. 2000; Gilpatrick et al.2000). Paramagnetic beads coupled to specific anti-bodies capture and concentrate the target viruses andallow the removal of potential inhibitory substances.

0944-5013/02/157/03-169 $15.00/0 Microbiol. Res. 157 (2002) 3 169

Microbiol. Res. (2002) 157, 169–175 (761)http://www.urbanfischer.de/journals/microbiolres

Detection of enteroviruses, hepatitis A virus and rotaviruses insewage by means of an immunomagnetic capture reverse trans-cription-PCR assay

Nerea Casas, Ester Suñén

Departamento de Inmunología, Microbiología y Parasitología, Facultad de Farmacia, Universidad del Pais Vasco, Apdo. 450,01080 Vitoria-Gasteiz, Spain

Accepted: March 23, 2002

Abstract

Corresponding author: E. Suñéne-mail : oipsupae@vc.ehu.es

The capture of intact antigenic and hence potentiallyinfectious virus particles on a solid phase followed byRT-PCR provides a better prediction of viral infectivityhelping to overcome this RT-PCR drawback. Moreover,IMC may be used in combination with a primary virusconcentration and purification procedure as a strategywith which to concentrate viruses from large volumesamples and improve the sensitivity of virus detectionby RT-PCR.

In this study, we describe a simple procedure for the enteric viruses detection by RT-PCR in a highly in-hibitory environmental sample such as sewage. In thisprocedure, simple preconcentration steps of the sampleare combined with a virus IMC from the concentratesand the detection of specific viruses by RT-PCR. Thesuitability of the procedure has been evaluated in fieldsamples for enteroviruses, hepatitis A virus (HAV) androtavirus detection.

Material and methods

Viruses and cell cultures. Poliovirus type 1 (PV1) strainLSc was propagated in Buffalo green monkey kidney(BGMK) cells and assayed for infectivity by the plaquetechnique (Sobsey et al. 1978). HAV, cytopathic strainHM175 was kindly provided by Dr. Sobsey from theUniversity of North Carolina at Chapel Hill. HAV waspropagated in FRhK-4 (foetal rhesus kidney-4 derived)cells and assayed by quantal assay estimating the mostprobable number of cytopathogenic units (MPNCU) per ml or g (Cromeans et al. 1987). The simian rotavirusstrain SA11 was grown and the titre in MA104 (rhesusmonkey kidney derived) cell line was determined byplaque technique (Bosch et al. 1988). Virus stocks wereobtained from infected cell lysates by freeze thawingthree times, followed by extraction with an equalvolume of chloroform. Human faecal suspensions con-taining rotavirus were chloroform extracted and used asa positive control for the IMC-RT-PCR.

Sewage samples. Raw sewage samples (1 L) werecollected from the metropolitan treatment plant ofVitoria, Spain. Samples were taken from November1999 to September 2000. Samples were collected in a sterile polypropylene container, kept at 4°C for lessthan 4 hours until their concentration and stored frozenat –80°C.

Concentration of viral particles. The method proposedby Shieh et al. (1995) with certain modifications was followed. Sewage samples were centrifuged at 4,000 × g for 30 min at 4°C to pellet solids. Thesediments were resuspended in 5 vol of 0.2 M glycine-0.15 M NaCl (pH 9.5), homogenized and centrifuged

at 4,000 × g for 30 min at 4°C. The supernatants werecombined and the pH adjusted at 7.2–7.5. Viruses in the supernatant were concentrated by precipitation with8% (w/v) polyethylene glycol 8000 (PEG) and 0.3 MNaCl at 4°C overnight. The precipitated viruses wererecovered by centrifugation at 13,000 × g for 30 min at 4°C and resuspended in about 16 ml of PBS-0.2%Tween 20, pH 7.4 per one litre of raw sewage. Sampleswere further purified by solvent extraction with an equalvolume of chloroform. Supernatants were recovered bycentrifugation at 2,000 × g for 30 min at 4°C. The finalconcentrates were analysed by RT-PCR directly afterheat release of viral RNA and after processing by IMCof the viruses. For seeding experiments, sample con-centrates were heat treated at 99°C for 2 min to destroyviral particles naturally present in the samples. The heat-treated samples were negative for PV-1 and HAVas evidence by IMC-RT-PCR testing.

Immunomagnetic Capture (IMC). Paramagnetic beadswith covalently linked goat antihuman IgG (BioMag®;Advanced Magnetics, Inc. Cambridge, MA) were usedin a 0.5 ml amount for each sample. The antibody coated beads were washed with PBS containing 0.05%Tween 80 to remove preservative buffer before beingused. The beads were blocked with 1% bovine serumalbumin (BSA) in PBS for 30 min at room temperatureand washed twice to remove the excess of blockingreagent. The beads were then suspended in 1 ml of PBS-0.1% BSA containing 0.8 mg / ml of a pool of IgGfrom human serum immunoglobulin (HSIG, GlobumanBerna P®; Instituto Berna, Madrid, Spain) and incu-bated, while rotating, for 30 min at room temperature.The antibody-beads complex was washed three timeswith PBS-0.05% Tween 80 to remove unbound anti-bodies. A volume of 500 µl of the sample concentratewas added. After incubating for a period of 2 hours withgentle mixing, the virus-beads complex was washedthree times with PBS-0,05% Tween 80. Following thefinal wash the beads were concentrated at the bottom ofa 0.5 ml tube by centrifugation at 7,000 × g for 2 minand the supernatant discarded. Then, the virus-beadcomplex was resuspended in about 50 µl of 0.1 × PCRbuffer II and incubated at 95°C for 5 min in order torelease the viral RNA. The beads were chilled on ice andpelleted by centrifugation at 13,000 × g for 2 min at4°C. The supernatant was analysed immediately by RT-PCR.

Some field samples (numbers 6, 7 and 8) were inhibi-tory for RT-PCR after IMC. In these samples, RNA fromvirus particles coupled to beads was extracted by usingPurescriptTM kit (PurescriptTM, Gentra, Minneapolis,USA) following the manufacturer’s protocol except that RNA was precipitated with isopropanol at –70°Cfor 1 h. Pellets were washed with ethanol, centrifuged at

170 Microbiol. Res. 157 (2002) 3

13,000 × g for 20 min and resuspended in RNA hydra-tation solution (Casas and Suñén, 2001).

PCR primers and oligoprobes. Nucleotide sequences ofprimers and internal oligoprobes for enteroviruses, HAVand rotaviruses have been described previously. Thehighly conserved 5’-end untranslated region of entero-viruses was used as the target for the synthesis of a pan-enterovirus 196-bp cDNA. For HAV, the genomicregion of the VP1 and VP3 junction was the target for a 192-bp cDNA (De Leon et al. 1990).

A nested RT-PCR was used for the detection of rota-viruses. Primers Beg9 and End9 amplified a 1,062-bpsequence from the VP7 gene of group A rotaviruses inthe first amplification reaction. Primers RV3 and RV4were used for a second amplification to obtain a 346-bpPCR amplicon (Gouvea et al. 1990; Le Guyader et al.1994).

RT-PCR. Reverse transcription was carried out withantisense primer at 42°C for 45 min by M-MLV reversetranscriptase (Perkin Elmer/Cetus, Norwalk, CT). Thereaction mixture contained 10 µl of the nucleic acidextract plus 16 mM (NH4)2SO4, 67 mM Tris-HCl (pH 8.8), 0.01% Tween 20, 5 mM MgCl2, 1 µM anti-sense primer, 200 µM of each deoxynucleotide triphos-phate (dNTP), M-MLV reverse transcriptase (2.5) andRNAase inhibitor (20 U) (Perkin Elmer). Amplificationwas performed in a final reaction volume of 50 µl con-taining 16 mM (NH4)2SO4, 67 mM Tris-HCl (pH 8.8),0.01% Tween-20, 2 mM MgCl2, 1 µM of each primer,200 µM dNTPs and 2 U of BiotaqTM DNA polymerase(Bioline, U.K.). For enteroviruses and HAV, a one step PCR reaction was used and the conditions of ampli-fication were as follows: an initial denaturation at 95°Cfor 5 min. followed by 40 cycles of 95°C for 1.5 min,55°C for 1 min and 72°C for 1.5 min and a final exten-sion at 72°C for 7 min. For rotaviruses, a nested PCRwas performed. The cycling conditions were 35 cycles,with each cycle consisting of 94°C for 1 min, 50°C for1 min and 72°C for 1 min. Nested PCR was performedwith 5 µl of the RT-PCR amplification product in a newbatch of PCR mixture. The amplification conditionswere 30 cycles as described before.

Undiluted and 1/10 dilutions of the nucleic acidextracts were analysed twice and negative controls (PBSused as samples) were added for IMC and RT-PCRexperiments. As positive controls of IMC and RT-PCR,the following viruses were tested: strain LSc of PV1,HM175 of HAV, human rotavirus from faecal suspen-sions and RNA from simian rotavirus SA11 (kindly pro-vided by Dr. Buesa).

Analysis of DNA by gel electrophoresis and hybridiza-tion. A 10–15 µl volume of amplified PCR products wasanalysed by electrophoresis in a 2% agarose gel, stained

with ethidium bromide and visualized through UV. Theamplified PCR products of enteroviruses and HAV weretransferred to a positively charged nylon membrane(Hybond-N+, Amersham, U.K.) by the Southern or slot blot methods. Nucleic acid was fixed by UV cross-linking for 5 min. Bound DNA was detected by hybridi-zation with 3’-end-labeled digoxigenin-dUTP oligo-probes. Oligoprobe hybridization and colorimetricimmunological detection of positive samples wereperformed by following the manufacturer’s instruc-tions (Boehringer Mannheim Biochemicals, Indianapo-lis, IN).

Results

Sensitivity study of IMC

The sensitivity of the IMC-RT-PCR procedure wasdetermined and compared to direct RT-PCR method byperforming assays with 10-fold serial dilutions of thevirus stocks in PBS. The detection limit was consideredthe highest virus dilution that demonstrated a positiveresult confirmed by oligoprobe hybridization. Thedetection limits were between 0.001 PFU and 0.01 forPV-1 and 0.1 MPNCU for HAV by using direct RT-PCRor IMC-RT-PCR. However, by using IMC-RT-PCRviruses can be detected in as much as a 1 ml volume ina microcentrifuge tube which increased the sensitivityof direct RT-PCR 100 times.

Concentration of viral particles from sewage samplesand IMC-RT-PCR analysis

Initially, a simple method consisting of an initial separa-tion of solids followed by polyethylen glycol precipi-tation and solvent extraction of the virus was evaluatedto concentrate and purify enteric viruses from sewagesamples to enable their detection by RT-PCR. Thisprocedure reduced sample volumes by about 65-fold;however, PCR inhibitors were not removed becausesample concentrates needed a 1-2 log dilution to give apositive signal in direct PCR (Fig. 1b and 2). A captureof viruses from the sample concentrates by using im-munomagnetic beads coupled to antibodies was as-sessed as a method of removing the remaining inhibitorsand to further reduce sample volumes. The efficiency of the IMC step was evaluated by seeding 500 µl ofheat-treated sewage concentrates (corresponding to30–35 ml of raw sewage) with different quantities ofPV-1 and/or HAV and processing the sample by IMCand RT-PCR. The IMC reduced the volume of sewageconcentrates by about 10-fold allowing the analysis of6–7 ml of sewage sample per RT-PCR reaction. TheIMC simultaneously removed inhibitory substances of

Microbiol. Res. 157 (2002) 3 171

amplification allowing the detection of enterovirusesand HAV in the sample concentrates without dilution(Fig. 1a and 2). The detection limits of IMC-RT-PCRfrom seeded sewage concentrates were between 0.1 and1 PFU for PV-1 and 1 MPNCU for HAV. The presenceand the amount of another virus did not affect the de-tection limit and the specificity of the IMC-RT-PCR.

Different combinations of concentrations of PV-1 (0.1to 103 PFU) and HAV (0.1 to 103 MPNCU) were subjectto IMC and RT-PCR and yielded consistent resultsregardless of the concentration of each virus (data notshown). Moreover, PV-1 and HAV were detected only inthe respective seeded samples using their specific pri-mers.

172 Microbiol. Res. 157 (2002) 3

Fig. 1. Comparison of IMC-RT-PCR (a) and direct RT-PCR (b) for detection of seeded poliovirus in concentrates of sewagesamples. Agarose gel electrophoresis of RT-PCR amplified products from concentrates seeded with 500 pfu of poliovirus. Lane M, DNA ladder; lane 1, undiluted sample; lanes 2–3, serial ten-fold sample dilutions; Lanes 4, positive control ; Lane 5,negative control. Arrows denote 196 bp enterovirus PCR products.

Fig. 2. Comparison of IMC-RT-PCR and direct RT-PCR for detection of seeded HAV in concentrates of sewage samples.Agarose gel electrophoresis of RT-PCR amplified products from concentrates seeded with 50 MNPCU of HAV. Lane M, DNAladder; Lanes 1–4, IMC-RT-PCR of HAV: undiluted sample (lane 1), ten-fold dilution (lane 2), positive control (lane 3) andnegative control (lane 4). Lanes 5–8, direct RT-PCR of HAV: undiluted sample (lane 5), ten-fold dilution (lane 6), positivecontrol (lane 7) and negative control (lane 8). Arrow denotes 192 bp HAV PCR products.

Field samples

Eight raw sewage samples were analysed for entero-viruses and HAV by IMC-RT-PCR and oligoprobehybridization and for rotavirus after a nested PCR.Enteroviruses were detected in 6 samples (75%), HAVin all analysed samples and rotaviruses in 6 samples(75%) (Table 1). The positive results for these samplescorresponded to an initial raw sewage volume of about6.5 ml. In three samples, (numbers 6, 7 and 8), PCRinterferences still remained after the IMC. In these sam-ples, the RT-PCR virus detection was possible after anRNA extraction by using a commercial system, Pure-scriptTM. A nested PCR was indispensable for the detec-tion of rotaviruses since none of the samples produced avisible band in the first amplification.

Sample concentrate volumes equivalent to 11–15 mlof raw sewage were examined for the presence of enter-oviruses by cell culture infectivity assay. Enteroviruseswere detected in 3 samples that were also positive byRT-PCR. Three of the samples showed a high toxicityfor the cells. Two additional sewage samples werecytophatic effect negative one of them being RT-PCRpositive.

Discussion

The application of RT-PCR for the detection of entericviruses in environmental samples is mainly limited bythe low concentration of viruses and the presence ofinhibitory substances. In the present study, a simpleprocedure was used to concentrate and purify entericviruses from sewage samples to be detected by RT-PCRdirectly after heat release of viral RNA. The procedurewas rapid and very simple, reducing sample volumes byabout 65-fold. However, it did not remove PCR inhibi-

tors because the dilution of seeded sample concentrateswas needed to obtain a positive signal in RT-PCR. Thelow sample size per RT-PCR reaction reduced the sen-sitivity of the procedure and false negative results mayhave been obtained when viruses were diluted beyondthe detection limit of the assay.

An IMC assay was evaluated as a final step to simul-taneously concentrate and purify enteric viruses fromsewage sample concentrates to be detected by RT-PCR.The sensitivity of IMC-RT-PCR was similar to the directRT-PCR method when virus stocks were analysed inPBS. However, the concentration of the sample duringthe IMC increases the sensitivity of the assay since ahigher sample volume can be analysed by RT-PCR re-action. The IMC step reduced the volume of the sewageconcentrates by about 10-fold. A higher sample volumereduction could have been possible by decreasing thefinal beads resupension volume. However, sufficientsample volume was needed in order to analyse differentviruses from the same IMC for independent RT-PCRreactions. The IMC simultaneously removed inhibitorysubstances of amplification, allowing the detection ofenteroviruses and HAV in the sample concentrateswithout dilution. Although the detection limits insewage concentrates were 10 to 100 fold less than inPBS, they were comparable to other reports for sewageand other environmental samples (Prévot et al. 1993;Monceyron and Grinde 1994; Tsai et al. 1994; Shieh et al. 1997). The immunomagnetic capture of intact anti-genic viral particles may explain the decreased sensi-tivity. The detection limit and the specificity of the IMC-RT-PCR were not affected by the presence oramount of another virus. Non-specific binding of viru-ses to uncoated or coated beads has been reported bysome investigators (Monceyron and Grinde 1994; Bida-wid et al. 2000; Gilpatrick et al. 2000). A blocking stepwith BSA was included in order to reduce them. How-

Microbiol. Res. 157 (2002) 3 173

Table 1. Detection of enteroviruses, HAV and rotaviruses in field sewage samples

Sample no Virus detection by IMC-RT-PCR* pfu count of and date Enterovirus Hepatitis A virus Rotavirus enterovirus†

1st amplification nested PCR

1, January – + – + ND2, February + + – + 130 PFU / ml3, March + + – + 10 PFU/ ml4, April + + – – ND5, May + + – + 10 PFU /ml6, June + + – – T7, July – + – + T8, September + + – + T

*+, positive; – negative† pfu count of enteroviruses per ml of sewage sample; pfu were obtained from a total sewage volume of 11 to 15 ml. ND, notdetected; T, toxic sample

ever, in this study non-specific binding could not bedetected since broadly polyclonal antisera were used tomake possible the simultaneous capture of differententeric viruses. Although the use of monoclonal anti-bodies can increase the specificity of the assay, it limitsthe application to a few specific viruses. A mixture ofmagnetic beads coated with specific antibodies to dif-ferent viruses could be an alternative. Whatever theprocedure, the specificity of the method will dependlargely on the primer set used in the RT-PCR. In thisstudy, PV-1 or HAV were detected only in the spikedconcentrates and when the specific primers for eachvirus were used.

The entire described procedure was successfullyapplied to field sewage samples for the detection ofenteroviruses, HAV and rotavirus by RT-PCR and oligo-probe hybridization. PCR interferences still remained insome samples after the IMC, probably due to variationsin sewage quality in different seasons. These sampleswere collected in the summer season and a high concen-tration of inhibitors and other substances are expectedbecause of higher temperatures and lower rainfall. AnRNA extraction by using a simple commercial systemRT-PCR removed residual inhibitors from these samplesallowing the RT-PCR virus detection.

Sample concentrate volumes of raw sewage wereexamined for the presence of enteroviruses by a cellculture infectivity assay. An inconsistent correlation be-tween the results of PCR and cell culture assays wereobserved in one sewage sample that was cytophaticeffect negative and RT-PCR positive. This fact has oftenbeen reported by other investigators (Shieh et al. 1997;Gantzer et al. 1998; Green and Lewis 1999). It is pos-sible that enteroviruses were in the sample althoughthey did not cause cytopathic effect on BGM cells ortheir levels were too low to be detected by the cell cul-ture assay. The amplification of nucleic acids of in-activated viruses is unlikely since only antigenic unda-maged virus particles can be captured by the antibodiesbound to the beads. Although IMC-RT-PCR does notdemonstrate the viral infectivity it provides a better pre-diction than other chemical and physical methods ofRNA extraction since it detects the antigen associatedwith the viral RNA.

The detection of enteric viruses in sewage samples byRT-PCR gives information about viruses that are cir-culating in the population. The high prevalence ofenteroviruses and rotavirus in sewage is consistent withstudies carried out by other authors (Bosch et al. 1988;Puig et al. 1994; Green and Lewis 1999). However, thepresence of HAV in all samples may suggest that HAVwas endemic in the community and it was shed con-tinuously. Further studies are required to determine the extension of HAV contamination in environmentalwaters over longer periods. The identification and

analysis of the viral strains could be a useful tool for the study of the molecular epidemiology of hepatitis Aand other enteric viral infections in a community.

Although more research is needed to improve virusrecovery and RT-PCR detection, the procedure de-scribed in this study can be useful for environmentalvirological studies. The method is relatively rapid andvery easy to perform and enables an efficient, sensitiveand specific detection of enteric viruses by RT-PCR inhighly inhibitory environmental samples.

Acknowledgements

This work was supported by funds from the University of Bas-que Country, Project Number UPV/EHU 093.125-EA075/98

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