DETECTION OF ENTEROVIRUSES AND HEPATITIS A VIRUS IN ENVIRONMENTAL SAMPLES BY GENE PROBES AND POLYMERASE CHAIN REACTION Ricardo De Leon1 Research Associate Carol Shieh2 ResearchAssociate Professor Ralph S. Baric3 Associate Professor Mark D. Sobsey1 Professor 1Department of Environmental Sciences and Engineering 3Department of Epidemiology School of Public Health University of North Carolina at Chapel Hill Chapel Hill, NC 27599 2Department of Pathology

TulaneMedical Center New Orleans, LA 70112 INTRODUCTION The documented evidence of outbreaks of enteric viral diseasebyconsumptionofcontaminatedwaterhas increased significantlyinthepastdecade (Craun, 1988). Reports of enteric virus isolations from drinking water sources and from finishedwaterprovide evidence for an increasedneed for periodic or perhaps even routine monitoring of these waters for viral contamination (Gerba and Rose, 1990). Entericviruseshavebeenisolatedonatleast37 occasions fromtreateddrinkingwaterintheunitedStatesandaround theworld (Bittonetal., 1985; GerbaandRose, 1990), some of which met quality standards for indicator bacteria and other parameters (Deetz et al., 1984; Gerba et al., 1984, 1985 and 1986). Unfortunately, the methods used for human enteric virus isolation from water rely on animal cell culture and are technically difficult, time consuming and relatively expensive to conduct. In addition, current indicator bacteria inadequately reflect the presence of waterborne enteric viral pathogens. It has been clearly establishedthatentericvirusesaremorepersistentinthe aquatic environment and more resistant to water and wastewater treatment processes, including disinfection, than are fecal indicator bacteria (HoffI 1986; LeongI 1983). Widespread and routine monitoring of wastewater effluents or raw and finished drinking water for human enteric viruses will require rapid, sensitive and inexpensive methods. New virus detection methods based on the use of gene probes (nucleic acid hybridization) and polymerase chain reaction (target nucleic acid in-vitro amplification) for detection of specific enteric viruses or closely related groups of viruses show considerable De Leon 833promise as inexpensive and rapid methods for virus detection in the environment. Gene probes and other nucleic acid techniques have recently been used for the detection of enteric viruses in environmental samples. Gene probe hybridization to genomic viral RNA has been reported to detect 2.5 X 10 5 physical particles of rotavirus SA-11 (Dimitrov et al., 1985), 10 4

physical particles of hepatitis A virus (Jiang et al., 1986), and 500-1000 PFU of rotavirus SA-11 and coxsackie B3 (De Leon, 1989; Shieh et al., 1990). Margolin et al., (1986) have reported the detection of 1-10 PFU of pOliovirus using probes with a specific activity of 2 X 10 9 CPM/ug of DNA. In general, RNA probes are 5-8 fold more sensitive than DNA probes (Jiang et al., 1987; Shieh et al., 1990; Dubrou et al., 1990). Although most reports on detection sensitivities are 100 fold less than a desired goal of 1-10 infectious units, several investigators have reported detection of enteric viruses in environmental samples. Margolin et al., (1986), using a cDNA probe to poliovirus, detected enteroviruses in environmental samples, and these samples also were enterovirus positive by a modified cell culture assay (De Leon and Gerba, 1985). In a later study, Margolin et al., (1989) used pOliovirus and hepatitis A gene probes to detect enteroviruses in groundwater. Jiang et al., (1986) have used cDNA probes to detect seeded hepatitis A in estuarine samples with a sensitivity of 10 particles and in a subsequent study they detected naturally occuring hepatitis A viruses in estuarine samples using ssRNA probes (Jiang et al., 1987). All virus detections in environmental samples by gene probes thus far reported have been made with radioactively labeled probes. Non-radioactive detection systems are approximately ten times less sensitive (Jiang et al., 1987) and thus not directly applicable for detecting the low number of viruses expected in environmental samples. However, the sensitivity of gene probes in environmental

samples can be increased by the use of other molecular techniques in conjunction with gene probe hybridization. Recent advances in nucleic acid detection by gene probes coupled with target nucleic acid amplification techniques such as polymerase chain reaction (PCR) offer exquisite sensitivity and detection specificity of enteric viruses (Shieh et al., 1990) and other microbial agents in the environment. Polymerase chain reaction (PCR) provides a 10 5 -10 6 enrichment of a specific nucleic acid sequence and is capable of detecting a single copy of DNA (Ou et al., 1988; Saiki et al., 1988) . Although Thermus aguaticus (Taq) polymerase cannot use RNA as a template, the PCR technique is adaptable to the amplification of RNA viruses by a simple reverse transcriptase reaction prior to polymerase

chain reaction (RT-PCR). Recently, this approach has been

used for the amplification of enteroviruses (Hyypia et al. , 1989; Rotbart, 1990; Chapman et al., 1990; Shieh et al., 1990), hepatitis A virus (Jansen et al., 1990) and rotaviruses (Gouvea and Glass, 1990). 834

### The choice of primers for PCR amplification of specific gene sequences in targetviral genomes is crucial for the efficiency and the specificity of themethod in detecting particularviruses and virus groups. Based on genetic and antigenicinformationitshouldbepossibletoselectthree setsofprimersthatwillmakeitpossibletodetectnearly all of thehuman enteroviruses, hepatitis Avirus and the group A rotaviruses. Some of the primers used for RT-PCR amplificationofenteroviruseswereselectedfromthe5'end non-coding region of their genomes. These regions contain highly conserved sequences that permit the development of pan-specific primers and probes capable of detecting a variety of different enteroviruses, including the polioviruses, group A and B coxsackieviruses and echoviruses (Tracy, 1988). Hepatitis Avirus is unrelated innucleotidesequencetootherenteroviruses, andseparate amplificationprimers were selected from both it's 5' end and specific capsid regions which are highly conserved among strains isolated from different parts of the world (Cohenet al., 1987; Gust and Feinstone, 1988). Because sequenceinformationforhumanrotavirusesismorelimited, primer selection was based on a commercially available primer set. Theobjectiveofthisstudywastodevelopandevaluatethe basic methodology needed for the detection of enteroviruses, hepatitis A virus and rotavirus in environmental samples by the combined use of reverse transcriptase-polymerase chain reaction and nonradioactively labeled oligoprobes. MATERIALS AND METHODS 

### VIRUSES AND CELLS EnteroviruseswerepropagatedinA549 (humanlungcarcinoma -derived) or BGMK (Buffalo greenmonkey kidney) cells and assayed for infectivity by plaque assay (Sobsey et aI, 1978). HAV, strainHM175, (NIHprototype strain) was grown in FRhK-4 (fetal rhesus kidney-derived) cells. other strains of HAV (CR326 and HAS 15) were propagated inA549 cells. Viruses were harvested from infected cell lysates byfreeze-thawing, Freonextractionandpolyethyleneglycol precipitation. HAV was further purified by equilibrium centrifugation in CsCl gradients and then rate-zonal centrifugation in sucrose gradients. Rotaviruses were propagated inMA-I04 (rhesusmonkeykidney) cells (Sato et al., 1981) andassayedfor infectivitybyplaqueassay (SAIl) or immunofluorescence (Human rotavirus Wa)(Smith and Gerba, 1982). PRIMERS AND PROBES Sequence analysis of several partial or fully sequenced enteroviruses indicates several highly conserved regions in the 5' end of the enterovirus genome (Iizuka et al., 1987; Jenkins et al., 1987; Tracy et al., 1985; Hughes et al., 1988; Pilipenko et al., 1989; Hughes et al., 1989; Toyoda et al., 1984). Some of these regions are also conserved in the closely-related rhinoviruses and bovine enteroviruses. Primers selected from conserved regions in

835human enteroviruses but not found in the rhinoviruses nor animal enteroviruses would be most appropiate for environmental samples. The downstream or antisense primer was selected from a region conserved in the human enteroviruses but lacking in the rhinoviruses and bovine enteroviruses which have been sequenced (Figure 1) (Jenkins r 1987; Earle r J.A.P. et al. r 1988). The downstream enterovirus primer (Figure 1) (EV-R 5' -ACCGGATGGCCAATCCAA3') r which was synthesized as single stranded DNA using an automated synthesizer is complementary to the sequences comprising nucleotides 627-644 in the coxsackie B virus genomic sequence (Jenkins r et al., 1987; Iizuka et al.,

1987). The upstream enterovirus primer (EV-L 5' CCTCCGGCCCCTGAATG-3') was synthesized in the (+) sense orientation from the sequence of the coxsackievirus B genome and comprises nucleotides 449-465. A strongly conserved region in the intervening sequences between the primers was chosen as an oligonucleotide probe. The probe comprises sequences 545-567 (EV-In 5'- ACTACTTTGGGTGTCCGTGTTTC- 3') of the coxsackievirus B genome and was synthsized in the (+) -sense orientation, thus capable of hybridizing only with cDNA or PCR product and not with viral genomic RNA. Primer selection for hepatitis A virus was based on the

HM175 strain. Two regions of the genome that exhibit high sequence conservation, the 5' non-coding sequences and the VP3 capsid protein, were used (Cohen 1987; Gust and Feinstone, 1988). The downstream 5' non-coding region primer for HAV (Table 1) (HAV-5 'R 5' -CGGCCAGAGCCTAGGGCAAG3') is complementary to the sequence comprising nucleotides 243-262 of HM175. The upstream primer for the 5' non-coding

region of HAV (HAV-5'L 5'-CCTCTTGGAAGTCCATGG-3') was synthesized in the (+) -sense orientation and comprises sequences 33-50 of the HM175 HAV strain. The internal probe for the resulting amplified fragment comprises sequences 117-134 (HAV 5'-In). The downstream primer in the capsid

region of HAV (HAV-CR 5'-CTCCAGAATCATCTCC-3') is the complementary sequence to nucleotides 2211-2226 of the HM175 strain and the upstream primer in the capsid region (HAV-CL 5' -ACAGGTATACAAAGTCAG-3') is in the (+) sense orientation. The internal probe comprises sequences 2170- 2191 (HAV C-In 5'-TTGCTCCTCTTTATCATGCTAT-3').

The rotavirus primers were obtained from a commercial supplier (GENEMED, Biotechnologies Inc., San Francisco CA

94080). The supplier derived the primers from segment 6 of the human rotavirus (HRV) Wa strain genomic sequence. The downstream primer (HRV-R 5'-ACAGACTTTCATTTGCGTCCGCAA-3') is complementary to sequences 1028-1051 and the upstream primer (HRV-L 5'-ATAGAAGACAGCGCACCGGATTTG-3' is homologous to sequences 451-474. The internal probe, also provided by GENEMED, comprises sequences 631-670 of HRV Wa (HRV-In 5'- CGAACATACAGCAATTTGAACATATCGTCCAGCTTAGGCG-3') (Both et al., 1984) . REVERSE TRANSCRIPTION AND ENZYMATIC AMPLIFICATION CRT-PCR) Procedures based on single tube reverse transcription and polymerase chain reactions were used preferentially. The 836methodofGouveaandGlass (2990) wastestedwiththethree virus groups: enteroviruses, HAV and rotaviruses. This method utilizes 2X Tag buffer for both the reverse transcriptionandtheenzymaticamplificationand includes 7% DMSO inthereaction. TagpolymeraseandTagbufferfrom Promega ( 2 XTagbuffer= 50 mM KCI, 20 mM Tris-HCI pH 9.0 at 25°C, 2.5

### roMMgCl2, 0.02% gelatinand 0.2% TritonX-200) were used with this method. The RNA-PCR kit from Perkin Elmer-Cetus (Norwalk, CT) was tested with polioviruses following manufacturer's instructions, with the exception that the reactionvolume during reverse transcriptionwas increased from 20 to 30 ul to accomodate a larger sample volume. The downstream primer was used during reverse transcription by the Gouvea and Glass method for both HAV and other enteroviruses. In the case of rotaviruses, cDNA synthesis was initiated with the upstream and downstream primerpairbecause rotaviruses have a dsRNA genome. When using the Perkin Elmer-Cetus RNA PCR kit, the random primers provided were used for the reverse transcription reaction. The reactioncocktailwasprepared ina singletube, mixed anddispensed into individual tubes for all sampleswithin a singletrial. The reaction cocktail consists of 20 ul of 20X tagbuffer, 20 ul of a 2.5

### roM nucleotidemixture, 7 ul of DMSO, 60 ul of deionized water, and 2 nanomoles of downstream primer. Each tube was overlayedwith 200 ul of mineral oil (Sigma, st. Louis, MO) and sample added underneaththeoil ina 20ulvolume. Thetubeswereheated at 99°C for 5 min inthethermal cycler, chilledon ice for 2min, centrifugedbriefly, andthen5 unitsofAMVreverse transcriptase and 20-20 units of RNasin were added underneath the oil. Samples were added to the thermal cyclerwitha preheatedblockat42°Candincubatedforone hour. After cDNA synthesis, sample tubes were heated to 99°C for 5 min to completely denature the reverse transcriptase. Aft·er brief centrifugation, additional primers and 5 units of Tag polymerase were added to each tube. Samples were amplified in a Perkin Elmer thermal cycler for 40 cycles. The cycle sequence was as follows: 95°C for 2.5 min, 49 or 55°C for 2.5 min, and 72°C for 2.5 min. Sampleswerekepton iceuntiltheblockreached95°C. OLIGOPROBES AND PCR PRODUCT HYBRIDIZATION Oligoprobes were 3 I labeled with digoxigenin-UTP using terminaltransferaseandpurifiedbyethanolprecipitation following instructions in the BMB Genius non-radioactive endlabellingkit (Indianapolis, IN). PCRreactionproducts (2.5-20 %of reaction volume) were mixed with 90 ul of 2 X SSC, boiled for 5 min, chilled on ice and slot-blotted on nylon membranes (DuPont, Boston, MA). The blotted samples were bound to the membranes by UV crosslinking using shortwaveUV light for 3 min ata distance of 25 cm. prehybridizationandhybridizationwereperformedfollowing BMB non-radioactive detection system specifications. Membranes were prehybridized for 4 hrs and hybridized overnightwith 5-20ng ofdigoxigeninlabeledoligomerper mL of solution. Theblotswerewashedtwicewith 6X SSC at

837room temperature and three times with 6X SSC at Tm-20oC of the oligo PCR product hybrid. ENVIRONMENTAL SAMPLE PROCESSING FOR PCR Eight liters of a raw sewage sample were concentrated**~OO**

fold by adsorption to an electropositive 30S filter and eluted with beef extract following conventional methodology (AWWA,**~990).** The sewage concentrate was fluorocarbon extracted, filter sterilized through a 0.2 um pore size filter (pretreated with beef extract) and seeded with an equivalent of 500 PFU of poliovirus for each subsequent PCR

reaction. The virus seeded raw sewage concentrate constituted 70% v/v of the final PCR reaction. Sephadex G200 (Pharmacia, Piscataway, NJ) spun columns were prepared according to Maniatis**(~982)** except that silane treated glass wool (Supelco, Bellafornte, PA) was used to plug the column and the centrifugal force was decreased to 400 x g to avoid crushing the column. Column volumes of**~.5** and 2.0 mL were used to process 0.25 mL of seeded sewage concentrate. Spun column excluded volume effluents were used directly for RT-PCR. RESULTS PRIMER SELECTION The detection of enteroviruses, hepatitis A virus and rotaviruses has been targeted because the human enteroviruses represent excellent candidate indicator viruses for fecally contaminated water and both HAV and rotaviruses are significant pathogens documented to be transmitted by drinking water. Sequence comparison of the 5' non-coding region (740 bases) of poliovirus typees**~,**

2 and 3 (Toyoda et al.,**~984),** coxsackie viruses A**2~** (Hughes et al.,**~989),** B**~,** 3 and 4 (Iizuka et al.,**~987;** Tracy et al.,**~985;** Jenkins et al.,**~987),** rhinovirus**B~** (Hughes et al.,**~988)** and a bovine enterovirus (Earle et al.,**~988)** indicate strongly conserved regions from which amplification primers and specific oligo-probes may be selected (Figure**~).** Strong sequence conservation at nucleotides 627-644 is observed between PV**~,** 2 and 3, CB**~** and 3 and**CA2~,** whereas the bovine enterovirus exhibits strong divergence in this region. Two other sections with strong sequence conservation within the picornaviruses can be readily identified at nucleotides 449-475 and 536-567 (Figure**~).** The upstream primer has been selected at sequences 449-465 and the internal probe at sequences 545- 567. The amplified segment is**~96** base pairs long. Previous studies indicate that the 5' non-coding and the VP3 capsid protein regions are highly conserved among different HAV strains (Cohen et al.,**~987** i Ticehurst,**~988).** To detect HAV, a primer pair from each conserved region was selected. The ability to amplify HAV from two regions could potentially be used as a confirmatory test. In the 5' non-coding region the downstream primer comprises nucleotides 243-262 and the upstream primer nucleotides 33- 50. In the VP3 capsid protein region nucleotides 2020-2037 and**22~~-2226** were used as a primer pair. Commercially 838available rotavirus primers derived from segment 6 of the Wa strainwere used (Table 1). RT-PCRAMPLIFICATIONOFENTEROVIRUSES. HAVANDROTAVIRUSES Previous work by our group (Shieh et ala I 1990) has demonstratedthatthepan-enterovirusprimerpairselected can amplify 17 of 17 enteroviruses tested (including poliovirus 1, coxsackieviruses A 5, 9, 12, and 17, coxsackieviruses B 3 and 4, echoviruses 1, 2, 4, 5, 7, and 12) when theprimer annealing temperature is kept at 45°C. Hepatitis Avirus was not amplifiedby these primers. The pan-enterovirus oligoprobe also detected all 17 enteroviruseswhenbothhybridizationandwashtemperatures were kept at Tm -20°C. Thepan-enterovirusprimerswereusedtoamplifypoliovirus 1 and coxsackievirus B3 under various conditions. The use of random prlmlng during reverse transcription was investigatedbecausethiscouldpotentiallyconvertallRNA viruses in an environmental sample into the more stable cDNA ina singlereaction. Reversetranscriptionprimedby random hexamers followed by PCR amplification resulted in some non-specific amplification of CB3 RNA extracted from cells if the annealing temperature during PCR was 45°C. When poliovirus 1 virions and a pan-entrovirus primer annealing temperature above 49°C were used, only the expected 196 bp band was observed, even after 40 cycles (Figures 2A and 3A). These pan-enterovirus primers worked effectively in amplification cocktails containing either no DMSO or7% DMSO. TwelvepercentDMSO worked only ifthe tubeswerenotoverlaidwithmineral oilduringthe initial heating of the sample. Amplification from CB3 cellularderivedRNAwasalsosuccesfulwhenallreactioncomponents were added before the RT reaction and then proceeded through PCR amplification with the omission of a 5 min heating at 99°C betweenRT and PCR amplification (data not shown)• Detection of HAV by RT-PCR with the 5' end non-coding region primer pairwas succesfull butmore difficult than forotherenteroviruses. Theuseof7% DMSO inthereaction cocktail was necesary and only moderate yields of PCR product were obtained (Figure 2A). The amplification products are not always "clean" and specificity of amplification varied among strains. HAV amplification was more successful from the VP3 capsidprotein. An amplified fragmentof207bpisreadilyobtainedwhenprimersderived fromnucleotides 2020-2037 and 2211-2226wereused (Figure 2A andTable 1). Comparisonsbetweenthe 5' non-codingand VP3 region primer sets indicate that the capsid region primersmayalsoresultingreatersensitivity (Figure2A)

. An amplified fragment ofabout 601 bp in length is readily obtained from the Wa strain of rotavirus using the commercial primer pair (Figure 2A). Nonetheless, no specificamplificationofthreegroupAanimal rotaviruses (the simian strain SA-11, the bovine strain NCDV-1 and a porcinerotavirusnatural isolate) and eightgroupAhuman isolates (2 each from the four known human serotypes) was 839**~~<\** obtained using the commercial rotavirus type A primer pair under the same conditions (Data not shown). RT-PCR of these viruses was unsuccessful even with a primer annealing temperature of Tm -20°C. When amplifying at the low annealing temperature,. a smaller, secondary band (approximately 220 bp long) can be observed with Wa rotavirus and with some of the human rotavirus isolates. The need to select alternate rotavirus primers which can amplify human rotaviruses and, hopefully, exclude animal rotaviruses is evident. SIMULTANEOUS AMPLIFICATION OF POLIOVIRUS r HEPATITIS A VIRUS AND ROTAVIRUS The amplification of enteroviruses, HAV and rotaviruses in a single PCR run is a practical approach to determine the presence of viruses in a sample. To test the feasibility of this approach, HRV Wa, PV1. and HAV were each amplified in a separate tube with their respective primer pair. RTPCR amplification of the three viruses was readily obtained

in a single PCR run when the lowest annealing temperature (49°C) of all the primer sets was used (Figure 2A, Table 1.). The expected amplified fragments were obtained for HRV

Wa (601. bp), PV1. (1.96 bp), HAV in the capsid region (207 bp) and HAV in the 5' non-coding region (230 bp). Although each virus was amplified in a separate tube, it is evident from these results that multiple viruses can be detected simultaneously. The amplification of multiple virus groups in a single tube reaction may also be feasible, but its effectiveness will have to be determined empirically. When an annealing temperature of 49°C is used to amplify HRV Wa,

a smaller secondary band is frequently obtained which is not observed when annealing temperatures are above 55°C. Single and distinct bands were obtained for PV1. and for HAV. Amplification from the 5' end of HAV was less efficient than in the capsid region. The selection of

multiple primer sets with similar Tm and non-complementary sequences and the optimization of RT-PCR conditions may lead to multiple virus group amplification in a single tube reaction. The PCR products obtained from the simultaneous amplification of HRV, PV1., and HAV were direct blotted (1./40th of the reaction products) in four replicate membranes. In addition to the amplified fragments, lambda DNA (250 ng per slot) as a non-specific probe binding control and 1.00ul of the diluent as a negative control were blotted. The negative control for the HRV amplification was blotted on the membrane to be probed by the respective HRV oligoprobe and so forth for the other 3 membranes. Also SA1.1. rotavirus segment 6 cDNA was blotted in the membrane to be probed by the HRV oligoprobe, coxsackievirus B5 5' end cDNA (nucleotides #1.-747) (Shieh et al., 1.990) as control

for the pan-enterovirus probe, and HAV 5' cDNA (nucleotides #1.62-848) (Shieh et al., 1990) as control for the 5' end HAV probed membrane. Each membrane was hybridized with a non-radioactively labeled HRV Wa, pan-enterovirus, HAV capsid, or HAV 5' internal oligomer at Tm -20°C (Figure 2B). The results of this experiment clearly indicate that each oligomer probe was highly specific and hybridized only 840tocDNA ortothe amplified fragment havingcomplementary sequences, butnottothenegativecontrols. TheonlycDNA controlwhichcouldbedetectedbyanyofthe four oligomer probes was the cDNA from the coxsackievirus B 5' end sequences #1-747. The Wa rotavirus probe has 12 base mismatcheswiththe equivalentregion inSA-11. The 5' HAV oligoprobe sequences (117-134) arenot included intheHAV cDNA blotted (162-848). These data clearly indicate that simultaneousdetectionofseveralvirusgroupsispossible. SENSITIVITY OF RT-PCR AND NON-RADIOACTIVE OLIGOPROBES The sensitivity of two RT-PCR procedures was determined with PV1. Themajor diff·erence between the two procedures tested is the reverse transcriptase reaction conditions. One procedure utilizes 1 X Tag buffer (low magnesium concentration), thedownstream (3') primer, and7% DMSO in a 100 ul reaction volume. This procedure resulted in detection of 0.5 PFU of poliovirus as determined by 

### uv visualizationofethidiumbromide-stainedgels (Figure3A)• A second RT-PCR procedure, with a comercial kit (Perkin Elmer-Cetus), has a high magnesium concentration buffer, random primers and an adjusted 30 ul reaction volume for thereversetranscriptionreaction. The sensitivityofthe RT-PCR commercial kit is 10 fold greaterthantheprevious method (0.05 PFU of poliovirus) (Figure 3A). Direct slot-blotting of 10 ul of RT-PCR reaction products fromthepoliovirus sensitivitydeterminations followedby non-radioactive, pan-entero, oligoprobe hybridization clearlydemonstratesspecifichybridizationtoprobeatthe same level of sensitivity as obtained byUV visualization of the ethidium bromide stained gel (also with 10 ul of reaction) (Figures 3A and B). Non-specific reactions were notobservedbetweenthepan-enteroprobeandPCRamplified fragments of HRV or HAV. In addition, non-specific hybridizationsignalswerenotobservedwithheatdenatured viralparticles (10 fold dilutions 500-.005 PFU) spoted on the samemembrane. Thepan-enterovirus oligoprobesequence ishomologoustoviralgenomicRNA, thus itonlyhybridizes with the complementary DNA sequences obtained in RT-PCR. The intensity of the signal observed in figure 3B was obtained in less than 2 hrs of colorimetric reaction. RTPCR amplification of target viral RNA followed by nonradioactive oligoprobe detection produces sharp end-point titrations conducive to most probable number (MPN) estimates of virus numbers in samples. ENVIRONMENTAL SAMPLE CLEAN UP FOR RT-PCR Simple and direct virus purification methods compatible with RT-PCR detection are needed for processing of environmental samples. Preliminary experiments in sample clean-up using Sephadex G-200 spun colUmns and divalent cation chelation by EGTA on a 100 fold raw sewage concentrateseededwith500PFU ofpoliovirusindicatethat this approach is very promising (Figure 4). No RT-PCR amplification was observed in the PV seeded concentrate whichreceivednotreatment (laneA) orRT-PCR in2

### mMEGTA (laneB). Passageofsamplethrougha 1.5mLG-200 Sephadex column (6:1 column to sample volume) and RT-PCR in 2 

### mM

841EGTA results in a PCR amplified fragment of the appropiate size (lane C). Passage of sample through a 2.0 mL Sephadex G-200 column (8:1 column to sample volume) without EGTA results in a large amount of non-specific amplification (lane D). The combined use of a 2.0 mL column and RT-PCR in 2 roM EGTA gave the desired PCR product as a single band (lane E). An overall 85% recovery of poliovirus, hepatitis A virus and rotavirus was obtained in the excluded volume from the Sephadex G-200 spun columns (data not shown). DISCUSSION RT-PCR linked to non-radioactive oligoprobes is a very

promising method for monitoring of drinking water for important human enteric viruses, including enteroviruses, HAV and human rotaviruses, because it offers exquisite sensitivity as well as specificity of virus detection. In order to be useful, the primers and the reaction conditions selected for RT-PCR of specific groups of enteric viruses

should: (a) give specific amplification of the target viruses, (b) detect these viruses at the level of sensitivity required, (c) and provide reproducible results. The selection of pan-enterovirus primers which amplify human enteroviruses and exclude hepatitis A viruses, animal enteroviruses, rhinoviruses and other members of the Picornaviridae family would be most useful for monitoring water for human fecal contamination. The pan-enterovirus primer set and perhaps the HAV capsid primers best fulfill

these criteria. The results obtained in this study suggest

that the pan-enterovirus primers used are good candidates

for detection of human enteroviruses in environmental samples. However, extensive sequence analysis, testing of a larger number of enteroviruses, more widespread crossreactivity studies with other enteric viruses and other microorganisms encountered in water are needed to fully assess the adequacy of these pan-enterovirus primers for use with environmental samples. Other investigators have also selected primers in the 5' non-coding region of

the picornavirus genome for the amplification of picornaviruses (Hyypia et al., , 1989) enteroviruses and rhinoviruses (Rotbart, 1990; Gama et al., 1989) and enteroviruses (Chapman et al. I 1990). The majority of these investigators have selected their downstream primers closer to the 5' end of the genome in regions of greater sequence conservation with rhinoviruses and bovine enteroviruses (approximate map positions #443-468 [Hyypia), 547-562 [Gama], 584-603 [Rotbart]) than our downstream primer. Rhinoviruses can be excluded from amplification by selecting the downstream primer (used for the initial reverse transcription reaction) within the last 100-120 nucleotides of the 5' non-coding region of the enteroviruses, because these sequences are not found in the

rhinoviruses (Tracy, 1988). The primers chosen by Chapman et al. (1990), which are within a few nucleotides from the primers we have selected, successfully amplified 40 of 41 enteroviruses and the amplified products were detected by an internal oligomer similar to ours. Echovirus 22 was the only member not amplified by Chapman et al., which provides further evidence to a previous suggestion that it may not 842

### belong to the enterovirus group (Rotbart et al., 1988). A compilation of the 41 enteroviruses tested by Chapman et al. and the 17 previously tested by our group results in 47distinctmembers (66% ofallenteroviruses) successfully tested in RT-PCR using similarprimers. Sequence data for HAV shows that the 5' non-coding region and the VP3 capsid region are highly conserved among diverseHAVstrains (Ticehurst, 1989i 

### Cohen,1987). Fromour initial evaluation, a primer set in the 5' non-coding region of the HAV genome (nucleotides 33-50 and 243-262) didnotworkveryeffectivelywithall strainsofHAV. Most likelythisisduetothepredictedRNAsecondarystructure between nucleotides 1-41 of the HAV genome and not to sequenceheterogeneity (Cohen et al., 1987). Selection of primers downstream of nucleotide 41 may lead to better amplification, but overall RT-PCR of HAV appears to be difficult in the 5' non-coding region {Jansen, 1990}. Amplification of HAV from the VP3 capsid region using primers for nucleotides 2020 to 2037 and 2211 to 2226 worked consistently. Thus, this region may be a better candidate fordevelopment ofRT-PCRmethodology foruseby water utilities. The amplification of rotaviruses with the commercial primers derived from segment 6 was successful, in our hands, onlywithHRVWa strainand failedto amplifyeight other human rotaviruses (2 of each serotype). Sequence information for rotavirus is still limited but segment 4 may be a better genome location for detection of human rotaviruses (Flores, 1989). A cDNA probe of segment four of rotavirus Wa has been used to detect rotaviruses in environmental samples, and it did not exhibit crossreactivity to SA-11, NCDV-1 and a porcine rotavirus isolate (De Leon, 1989). Kantharidis et al., (1987) found significant sequence variation in segments 4 of human rotavirus strain RV-5 and simian rotavirus SA-11. In addition gene 4 may be used to distinguish between more virulent strains of human rotavirus from those which may produce asymptomatic infections (Taniguchi et al., 1989). Single tube RT-PCR methods differ in buffer composition, typeofreversetranscriptase, primerusedforfirststrand cDNA synthesis, and volume of the RT reaction. Of the two methods we tested, the commercial RT-PCR kit resulted in a ten-foldgreatersensitivityindetectionthana generic cocktail, witha positiveresult for as littleas 0.05 PFU of pOliovirus (approximately 5-50 molecules of RNA). The kit has been optimized for the reverse transcription reactionbyuse of highermagnesium concentration, random primers and smaller reaction volume (20 ul vs 100ul). However, for testing of environmental samples the volume of the RT reaction may need to be increased and the potential impact of this increased volume on the overall sensitivity of the assay must be investigated in future studies. The non-radioactive oligoprobe labeling and detection systemused in this study provides the needed sensitivity 843for detecting PCR products. strong colorimetric reactions were observed within one hour after the probe was hybridized to the target sequences and the digoxigenin specific antibody was bound to the probe. At the limit of sensitivity of virus detection by the RT-PCR methods used, the non-radioactive probe provided detection within one hour at the titration end point detected by gel electrophoresis of PCR products. Our preliminary investigations of environmental sample clean-up methods indicate that gel chromatography can be

adapted to the removal of RT-PCR-interfering factors. So far, only one type and grade of gel filtration material (Sephadex G-200) has been tested. clearly, a more thorough and systematic evaluation of other gel chromatography materials and grades, their combinations, optimization of gel bed volume to sample ratios, equilibration and running buffers, and potential divalent ion chelators is necesary in order to obtain an optimum clean-up method for routine use on environmental samples. Such investigations are proceding in this laboratory. The RT-PCR-oligoprobe method is a very promising non-conventional approach to the detection of enteric viruses in the envinronment. REFERENCES Bitton, G., Farrah, S.R., and Montague, C., Binford, M.W., Scheverman, P.R., and Watson, A. Survey of Virus Isolation Data from Environmental Samples, EPA Contract No 68-03- 3196, 1985, Cincinnati, OH. Chapman, N.M., Tracy, S., Gauntt, C.J., and Fortmueller, U. 1990. Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization. J. Clin. Microbiol. 28:843-850. Cohen, J.I., Ticehurst, J.R., Purcell, R.H., Buckler-White, A., and Baroudy, B.M. 1987. Complete nucleotide sequence of wild-type hepatitis A virus: Comparison of different strains of hepatitis A virus and other picornaviruses.**~** Virol 61:50-59. Craun, G.F. Surface Water Supplies and Health, Journal of American Water Works Association, 1988, 80: 40-52. Deetz, T.R., Smith, E.M., Goyal, S.M., Gerba, C.P., Vollet, JJ., Tsai, L., DuPont, H.L., and Keswick, B.H. 1984. Occurrence of Rota- and Enterovirus in drinking water and environmental water in a developing nation. Wat. Res. 18:567-571. De Leon, R. and Gerba, C.P. Enhancement of the Sensitivity of the BGM Cell Line to Enteroviruses, ASM Meeting, 1985, Washington, DC. De Leon, R., Use of Gene Probes and an Amplification Method for the Detection of Rotaviruses in Water, PH. D. Dissertation, University of Arizona, 1989, Tucson, AZ. 844 IDimitrov, D. 

### J., Graham, D.Y., and Estes, M.K., Detection ofRotavirusbyNucleicAcidHybridizationwith Cloned DNA of Simian Rotavirus SA-II Genes, Journal of Infectious Diseases, 1985, 152:293-300. Dubrou, S., Kopecka, H., Lopez-pila, J.M., Marechal, J., and Prevot, J., Detection of Hepatitis A Virus and other Enteroviruses inwastewater Samples by Gene Probe Assays, International svmposhim on Health-Relat.ed water Microbiology, 1990, Tubingen, Germany. Gama, R.E., Horsnell, P.R., Hughes, P.J., North, C., Bruce, C.B., Al-Nakib, W., and Stanway, G. 1989. Amplification of rhinovirus specific nucleic acid from clinical samples usingthepolymerasechainreaction. J. Med. Virol. 28:73- 77. Gerba, C.P., Keswick, B.H., DuPont, H.L. and Fields, H. A. 1984. Isolation of rotaviruses and hepatitis A virus from drinking water. Monogr. Virol. 15:119-125. Gerba, C.P., Rose, J. R. and singh, S.N. 1985. Waterborne gastroenteritis and viral hepatitis. CRC. crit. Rev. Environ. Control 15:213-236. Gerba, C.P., Rose, J.B., De Leon, R., Toranzos, G.A., Singh, S.N. and Keswick, B.H. 1986. Isolation ofrota- and entero- viruses from three different drinking water supplies. ProceedingsWaterQualityTechnologyConference. Houston. TX. AWWA. pp. 451-459. Gerba, C.P. and Rose, J.B. Viruses in Source and Drinking Water, in Drinking water Microbiology, Springer-Verlag, New York, 1990, pp. 380-395. Gouvea, V., Glass, R.I., Woods, P., Taniguchi, K., Clark, H.F., Forrester, B., andFang, Z.Y., 1990. PolymeraseChain ReactionAmplificationandTypingofRotavirusNucleicAcid from Stool Specimens, Journal of Clinical Microbiology, 28:276-282. Gust, I.D. and Feinstone, S.M. 1988. Hepatitis A. CRC Press, Boca Raton, FL. Hoff, J.C. 1986. Inactivation of Microbial Chemical Desinfectants. EPA/600/2-86/067, cincinnati, OH. Agents bv U.S.EPA,
Hyypia, T., Auvinen, P., andMaaronen, M. 1989. Polymerase chain reaction of human picornaviruses. J. Gen. Virol. 70:3261-3268. Jansen, R. 1990. Personal communication. Jlang, X., Estes, M.K., Metcalf, T.G., andMelnick, J.L., 1986. Detection of Hepatitis A Virus in Seeded Estuarine Samples by Hybridizationwith cDNA Probes, Appl. Environ. Microbiol., 52:711-717.

845Jiang, X., Estes, M.K., and Metcalf, T.G., 1987. Detection of Hepatitis A Virus by Hybridization with Single-Stranded RNA Probes, Appl. Environm. Microbiol., 53:2487-2495. Leong, L.Y.C. 1983. Review: removal and inactivation of viruses by treatment processes for potable water and wastewater. wat. Sci. Technol. 15:91-114. Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory. Margolin, A.B., Hewlett, M.J., and Gerba, C.P. 1986. Use of a cDNA Dot-Blot Hybridization Technique for Detection of Enteroviruses in Water, Advances in Water Analysis and Treatment. AWWA Proceedings, Houston, TX.

Margolin, A.B., Richardson K.J., De Leon, R., and Gerba, C.P. 1989. Application of Gene Probes to the Detection of Enteroviruses in water, in Biohazards of Drinking Water Treatment, Ed., R.A. Larson, Lewis Publishers, Chelsea, MI. Ou, C.Y. et al., science, 1988, 239:295-297. Rotbart, H.A. 1990. Enzymatic amplification of the enteroviruses. J. Clin. Microbiol. 28:438-442. Saiki, R.K., et al., science, 1988, 239:487-491.

sato, K., Inaba, Y., Shinozaki, R., Fujii, R., and Matamuto, M. 1981. Isolation of human rotaviruses in cell cultures. Brief report. Arch. Virol. 69:155-160. Shieh, Y.S.C., Baric, R.S., Sobsey, M.D., Ticehurst, J., De Leon, R., and Walter, R., 1990. Detection of Hepatitis

A Virus and other Enteroviruses in Water by ssRNA Probes, J. Virol. Methods, accepted for publication. Smith, E. M. and Gerba, C. P. 1982. Development of a method for detection of human rotavirus in water and sewage. Appl. Environ. Microbiol. 43:1440-1450. Sobsey, M.D., Shields, P.A., Hauchman, F.S., Davis, A.L., Rullman, V.A., and Bosch, A. 1988. Survival and persistence of hepatitis A virus in environmental samples. In Viral Hepatitis and Liver Disease, A.J. Zuckerman (ed.). pp. 121- 124. Alan R. Liss. Inc., New York. Ticehurst, J., Cohen, J.I. and Purcell, R.H. 1988. Analysis of molecular sequences demonstrate that hepatitis A virus (HAV) is a unique picornavirus. In: Viral Hepatitis and Liver Disease, A. Zuckerman (ed.). Pp 33-35. Alan R. Liss, New York. Tracy, S. 1988. The genome of the group B coxsackieviruses. In: Coxsackieviruses. A general Update, Eds. M. Bendinelli and H. Friedman. Plenum Press, New York. AWWA. 1990. Detection of Enteric Viruses, in Standard 846Methods for the Examination of Water and Wastewater, 17th Edition Supplement, 1990.

TABLE 1. PRIMERS FOR ENTERIC VIRUS AMPLIFICATION BY PCR

Amplified Primer Se@ence TmoC* Fragment Rotavirus Wa Segment 6 451-474 ATAGAAGACAGCGCACCGGATTTG 72 601bp 1028-1051 ACAGACTTTCATTTGCGTCCGCAA 70

Pan-enterovirus 5 INon-coding Region 449-465 CCTCCGGCCCCTGAATG 58 196bp 627-644 ACCGGATGGCCAATCCAA 56

HeQatitis A virus HM175 CaQsid Region 2020-2037 ACAGGTATACAAAGTCAG 50 207bp 2211-2226 CTCCAGAATCATCTCC 48

5 INon-coding Region 33-50 CCTCTTGGAAGTCCATGG 56 230bp 243-262 CGGCCAGAGCCTAGGGCAAG 64



## * Tm calculatedby adding 4°C for every Gor C and 2°C for everyA orT. The Tm of the rotavirus primers was provided by the supplier.

847449**46~)** 5 • - CGTCCGGCCCCTGAATG - 3' 445 502 AAGAATCG1'ccqGCCCCTGAA.l'GCGGC1'AATCCCI\ACCTCG(;GGC.I\GG,[,(;(;TC i\CAI\ACC ----G----- --------------- --- --------T- -- - f\---- AA- -- - -C
- -- -G --(;--

--- -- ---GI: ------- -------------------- T- T - - - -AT -.- .1\.-- --·-CA-·CTG- - ·-CGG1'-- - - - -- - - - -. - - - - - - - .. -.- - - - - - - - .- - -- .- T - -- --**Tl~** - --A- - - CA -Ace TGGT-G------- -- - --- -- - -------- ------- ----- -T -- -- TG- ---- -- --CACACCC1'- - - G-- ----G----------------------------T----A---A---ATC-C----G-C--**TT--G-------------------------C-TT--.I\-CT-.I\-C--T-GCTCAT~----­** GG1'TT--------AG-- ,,------1'---------

---- --C-A--G1'·- --CG----- T-- 505 (j62 AG1'GAT1'GGCCTGTCG1'AACGCGGAAG1'CCGTGGGGGAACCGAc;.:rAt..!'(l"['t;(:Gn;TCCGl'- ------C--- .--1'- -- - --- -- ----------T--- --- .. --------- - --- - ---.- --.- -- .------. --- --CAGCCA------------------------------------------------- --G-GGCA-1'G----------G----C--1'-CA------------------ ------- - --A-GGCA-1'G------ --- --G---- -C- --T -(;.1\-------

-- --- - ---.---
-- -- - --- -- ---- - -. -----GGT1'1: I: * -------T---1'------T---- ---- --------- -----. - --.--- -. -- .. -A1'--GCTTATG----- --T-A---- T-G- -G-AT---G------- --- .- ... --.- - -- - --.- - - - --- ----1'-GCTACG1'I:--- -----------TG--A---------A --- --- - ---C----.I\"'---- 565**b~~ G1'T_TGCT1'T1'A1'1'1'1'&A1'1'GTGGCTGCT1'ATGG1'GACAA1'CACAG.I\TTGTTAT** **\"~I\TAi\AG** -------G-------T--CA----------------------G-------------"--- -- ------ - -- ----C- TGA 11: A--- ---------------- -- --- T-- -- .--- -.- -. -- --- -- -. - .- -----1\-- - -----CC1'-- AG-- - ---.--- - - -- -- -- ---- -.-- TGACAGG - - - -. -·C -1'-- -----A--------cc* --AC----- -- --- ------ --------- TGAGI\ F_

- ---.- c- - - - T- ------C-----I\--C--I\C--------------------1'1'I\CGI\I\" -----c -1'-. -----AC---T-CC1'T1'A--G'" AAT1'-- -- --- - .----- -- -- ·-ATATACI\TI\l;.I\TATATATT

- --------A-1'I\--1'1' --ACI\ACI\A "'T - -.
---- --- - -- --T -G-- T -- -AG-AT -G - GTTC . 625 659 CGAA1'1'GGA1'1'GGCCA1'CCGGTGI\AI\G1'GI\"GATTCA 3' ['VJ ------------------ --------G1'- --TGT- F -CAG 3' PV2 -- -G------------ ----A----TG-I\-C-- - -- -A- 3' PV:5 &1'1'------------- --·-----·-CTAACI\" ---G(;I\- 3' CEIl**-T1I:~--------------------CCAAT---GCT--** 3' C83 - 1'& --------- ---- -C--I\-- - --CTGTGCAAT- --ATT 3'**CA~~1** GC--TCI\1'-GGGT-·- --CAGGTA1'C1' -G-CI\-AATG -1'G 3' R 18 -CC"--- a ----AAT--TGCC1'--GT-T-ACCTTI\-I\G 3' SF-V

627 644 3'-AACGl'AACCGG1'AGGCCA-S' FIGURE 1_ Sequence CompRrisons among Enteroviruser-;_ Sequences were aligned to the Sabin poliovirus type genomic sequence (Toyota, et aJ, J 984) . Hegj OIlS of homol**O[~y** are designated by dashed lines while astericks represent either nucleotide insertjons or delet.ions_ PVI-poti.ovirusJ, PV2-poliovirus 2, PV3-poliovirus 3, C81-coKsacki0 virus Bl,CB3-coxsackie virus [U, CA?l·-coxsackie 1\ 21, RBIrhinovirus 1, BEV-bovine enterovirus_ 848

### Figure 2A MultiplevirusRT-PCR. Eachvirusgrouphasbeen amplified with its respective primer pair in a separate reaction. Reverse transcription took place in 1 X Tag buffer from Promega, 7% DMSO and the 3' primer (see text). M= markers corresponding to 587,540, 504, 458, 434, 267, 234, 213, 192, 184, 124, 123 (BMB DNA marker V); HRV Wa= human rotavirus Wa strain; (-)= complete reaction cocktail without virus; PVl= pOliovirus type Ii 

### HAVcap.= hepatitis A virus amplified in the capsid regioni 

### HAV5'= hepatitis A virus amplified in the 5' non-coding region. The amplified fragment sizes are: HRV Wa, 601 bPi 

### PVl, 196bPI HAVcap, 207 bp and HAV5', 230 bp long.

849oligo probe HRVwa Pan-En HAVe HAVS'

RT- peR > 5t; cL > <: < .- a... I""'" I ,I Figure 2B

« zz 00"": -< V -0 Direct blotting of PCR products and non-radioactive detection by oligoprobe hybridization. The four membranes were blotted with 1/40th of the PCR products per slot obtained from the multiple virus PCR reaction and with

various controls. Each membrane was hybridized at Tm -20 0 C with either a rotavirus, pan-enterovirus, hepatitis A capsid region or hepatitis A 5'non-coding region internal digoxigenin labeled oligoprobe. HRV= human rotavirus Wa RTPCR amplified product; PV= poliovirus amplified product; HAVc= hepatitis A capsid region amplified product; HAV5'= hepatitis A 5' non-coding region amplified product; (-)= 10 ul of negative control; X DNA= 250 ng of lambda DNA; cDNA= for the HRV Wa oligoprobed membrane, 100 ng of SA-II segment 6; for the pan-enterovirus probed membrane, 100 ng of 5' end cDNA (bp 1-740), for HAVc, no cDNA spotted; for HAV5', 100 ng of HAV5' cDNA (bp #162-848). 850 jFigure 3A Sensitivity of RT-PCR for Poliovirus 1. M= markersi PFU PVl= 500-0.005 plaque forming units of poliovirus Ii (-)= complete reaction cocktail without virus. The virus dilutions in the left side of the figure were reversed transcribed in 1 X Promega Taq buffer and the downstream primer (3'Pr). Thevirusdilutions intheright sideofthe figurewerereversedtranscribedwiththePerkin-Elmer (PE) RT-PCR kit and random primers (Rp).

851RT-PCR Q.. Q.. MO c.t: COVl PFU > w.J**CJ~** PV1 0- 0-**~O** 500 - ........

SO - -

5 - - .5 ...... - .05 - .005 (- ) I , HRV HAV PCR product Figure 3B. sensitivity of RT-PCR and non-radioactive oligoprobe hybridization. Poliovirus RT-PCR-derived products were direct blotted (10ul from each dilution) and hybridized with digoxigenin-Iabeled pan-entero oligoprobe at Tm-20 C. PVl= pOliovirus 1 RNA from 500-0.005 PFU; PE Rp= amplified products from the amplification of pOliovirus with the Perkin Elmer RNA PCR kit using random primers; Tag B 3'Pr

DMSO= amplified products from the amplification of poliovirus with 1 X Promega Tag buffer, 7% DMSO and the downstream primer. HRV HAV PCR products= 10 ul of PCR

amplified fragments of human rotavirus Wa and hepatitis A VP3 capsid region, respectively. 852Figure 4. Detection of seeded poliovirus 1 in a raw sewage concentratebyRT-PCR. Each PCR reactioncontained500 PFU ofpoliovirus. The sewage concentratecomprised 70% vjv of the RT-PCR reaction volume. Lane A= no sample treatment; lane B= RT-PCR reaction cocktail with 2 mM EGTA; lane C= samplerunthrougha 1.5mLG-200 Sephadexspuncolumn (6:1 column to sample volume) and RT-PCR reaction with 2 roM EGTA; lane D= sample run through a 2.0 mL G-200 Sephadex column (8:1 column to sample volume) before RT-PCR; lane E= sample run through a 2.0 mL G-200 Sephadex spun column and the RT-PCR reaction cocktail contained 2 mM EGTA.

853