Environ. Sci. Techno/. 1995,29, 2636-2638
Infectious Enteric Viruses in ROSA M. PINTO, RODRIGO GAJARDO, F. XAVIER ABAD, A N D ALBERT BOSCH* Department of Microbiology. University of Barcelona, 08028 Barcelona, Spain
A procedure based on the infection of CaCo-2 cells and molecular hybridization with specific cDNA probes has been developed for the detection of infectious fastidious enteric viruses in environmental samples. CaCo-2 cells, derived from a human colon adenocarcinoma, showed an increased sensitivity when compared to the usual routine host cell line to laboratory strains of rotavirus 3, reovirus 3, astrovirus 1, poliovirus 1, coxsackievirus A 24, enterovirus 70, and adenovirus 5,40, and 41. Using this methodology, wildtype rotaviruses, enteric adenoviruses, enteroviruses, and for the first time, astroviruses have been detected in freshwater samples. Direct dot-blot hybridization alone was not sufficient for virus detection from environmental samples. CaCo-2 cells may be used as a universal in vivoamplification system for human enteric viruses, enabling the specific monitoring of infectious viral agents in the environment.
Introduction The limited sensitivity for some enteric viruses of conventional tissue culture methods have prompted investigators to search for novel procedures, such as molecular techniques, for the detection of viral-specific nucleic acid sequences. The use of gene probes for virus diagnostic in clinical samples has been widely described (1-3). In the case of environmental water samples, the use of molecular hybridization techniques has also been reported 14, 3 , although the sensitivity of the method is not high enough to detect the low number of viral particles sometimes present in such samples. An alternate and more sensitive method for viral detection is the amplification of viral nucleic acids by the polymerase chain reaction (s) whose application to environmental samples is increasing ever since (7,s).However procedures based on the use of gene probes or polymerase chain reaction amplification have to face the drawback that they do not differentiate between infectious and noninfectious particles. Amplification of virus sequences in cell culture prior to detection by gene * Corresponding author address: Department of Microbiology, University of Barcelona, School of Biology, Av. Diagonal 645, 08028 Barcelona, Spain; telephone: 402.14.85; Fax: 4 1 1.05.92; e-mail address:
[email protected].
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probe hybridization accomplishes the dual purpose of increasing the number of copies of target nucleic acid and of incorporating an infectivity assay as well. This has led to the search for continuous cell lines susceptible to the infection of a wide range of enteric viruses. The studies described herein show the use of the continuous cell line CaCo-2 combined with molecular hybridization with specific probes to perform infectivity assays of several fastidious enteric virus strains present in water samples.
Materials and Methods The following virus laboratory strains have been used throughout these studies: human adenovirus type 5 (ATCC VR-5; Ad5), type 40 (Hovi-X Ad401, and type 41 (26341-77; Ad41),groupAhumanrotavirusserotype3 (ItoIP13;HRV3), poliovirus type 1 (LSc 2ab; PVl), hepatitis A virus (HM175; HAW, human reovirus type 3 (Re03),enterovirus 70 (E70), and coxsackievirus A24 (CA24). Ad5 was propagated in Hep2 cells, Ad40 andAd4l were propagated in PLClPRFl5 cells, HRV3 and Re03 were propagated in MA104 cells, HAV was propagated in FRhK-4 cells, and PV1, E70 and CA24 were propagated in BGM cells using standard procedures. CaCo-2 cells were cultivated in Eagle's minimal essential medium (MEM) supplemented with 0.03% glutamine, 0.075% sodium bicarbonate, 100 U of penicillinlml, 100 pg of streptomycinlml, and 10%fetal bovine serum (FBS). CaCo-2 cells were passaged at weekly intervals (split ratio 1:3) and used at passage level 80-100. Viral enumerations were performed by calculating the most probable number of cytopathogenic units per milliliter (MPNCUlmL) by infecting cell monolayers grown in 96-wellmicrotiter plates, as described elsewhere (9). Briefly, confluent monolayers of cells were incubated overnight in MEM without FBS. Virus stocks were preactivated for 30 min with 10 pg/mL of trypsin (1:250 tissue culture grade; Difco, Detroit, Mi) at 37 "C, serially diluted 10-fold, and inoculated onto cells. After 45-min adsorption at 37 "C, 100 p L of serum-free maintenance medium and with 5 pglmL of trypsin were added. Plates were checked daily for cytopathic effect (CPE). Wild-type enteric viruses were concentrated on location from 500 L of fresh water by filtration through Zeta Plus MK I1 filters (AMF Cuno), elution with 500 mL of 3% beef extract-0.05 M glycine buffer, and organic flocculation to a final volume of 50 mL, followingthe procedures described elsewhere (10). Fastidious wild-type enteric viruses in water samples were assayed by dot-blot hybridization before and after infection of CaCo-2 cell monolayers, using specific cDNA probes. Extraction of viral nucleic acids from 0.1 mL of concentrated water samples was performed as described elsewhere (I,2,10). Confluent monolayers of CaCo-2 cells in T25 flasks were inoculated with 0.2 mL of a 1 : l dilution of trypsin-treated concentrated water samples. After adsorption, 10mLof serum-free maintenance medium with 5 pg/mL of trypsin were added. At 48 h postinfection (pi.), RNA or DNA was extracted from the cultures for the determination of viral growth. Briefly, for adenovirus detection, DNA was extracted from RNase-treated infected cell lysates by heating at 60 "C for 30 min in the presence of 100pglmL of proteinase K, followed by phenol extraction (pH 8.0) and ethanol precipitation. For rota-, entero- and astrovirus detection, the same procedure was essentially
0013-936~95/0929-2636$09.00/00 1995 American Chemical Society
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FIGURE 1. Compantive titration of enteric viruses in CaCo-2 cells (filledcircle)and in the convenlionallyused cell line to reachvirus lopen circle) IBGM tor PV1. MA104 tor HRV3 and Red. HepZ for Ad40 and Ad41. FRhK-4for HAVI.
followed, although RNA was extracted from infected cells insteadof DNA, avoidingRNase treatment and usingwaterequilibrated phenol (pH 5.21. Probes47B (1. Flores, personal communicationl. 21T43 (111, N26 (21, M9 (21. and PV104 (121 were used to detect rotaviruses, astroviruses, Ad40, Ad41, and enteroviruses, respectively. For astrovirus, two additional blind passages (0.1 mL) were performed in CaCo-2 cells, and RNA extraction and nucleic acid detection by gene probe were attempted after each cell passage. Probes were obtained and labeled with digoxigenin-llUTP (Boehringer Mannheim. Barcelona), following the manufacturer's instructions. Adot-blot hybridization assay was performed with the different cDNA probes following standard procedures. Prehybridization and hybridization (50 ng of probelmL of solution1 were carried out at 42 "C in the presence of 50% formamide. Positive signals were detected by the use of a photochemical signal generating system (Boehringer Mannheiml.
Results To ascertain the validity of the methodology described herein, comparative quantitations of stock suspensions of different enteric virus strains were performed in CaCo-2 cells and in the conventional cell line for each viral strain. The onset of CPE appeared earlier in CaCo-2 cells than in the standard cell line for HRV3, Ad5. Ad40, Ad41, Re03, E70, and CA24. appeared at the same time as in BGM cells for PVI. and appeared later than FRhK-4 for HAV. Virus titers obtained in CaCo-2 cells after 1 day pi. were consistently higher than when using any other cell line. At 7daysp.i.. titersinCaCo-2 cellsalwaysequaledorexceeded virus numbers obtained in other cell lines, with the sole exception of HAV. which gave the highest titers in FRhK-4 cells. The comparative titration of some of these virus strains in CaCo-2 cells and in the cell line regularly used for each virus is shown in Figure 1. The methodology was applied to detect wild-type fastidious enteric viruses in concentrated water samples collected from sites receiving sewage pollution. Specific nucleic acids from rotaviruses, astroviruses, adenoviruses. and enteroviruses were not detected in any of six different concentrated water samples (Figure 21. However, after infection of CaCo-2 cells, three of these samples were positive for enterovirus, two were positive for rotavirus, and onewas positiveforAd40 (Figure2). Afterone passage
FIGURE 2 Dot-blot hybridization analpis showing ampliicstion of nucleic ecids from wild-typeememvirus. rotavirus. and adenovirus 00 in water samples aher infeclion of CaCo.2 cells. Columns trom leh IO right probe posilive and negative controls; 1 6. samples: A, nonamplified samples; B. amplified samples.
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FIGURE 3. Dot-blot hybridization analpis showing ampliiication of astrovirusnucleic acid lrom water samplesby successive passages in CaCo-2 cells. Columns from Ish Io right: probe positive and negative controls; 1 and 2. samples: A, lint passage; B. second passage: C, lhird passage.
in CaCo-2 cells, specific astrovirus nucleic acids were not detected in any of the six different concentrated water samples. Since thesensitivityoftheastrovirusprobe21T43 appears to be very low (9).two samples positive for other viruses were blind-passaged two more times in the CaCo-2 cells to amplify the target nucleic acid and to increase the chances to detect astroviruses. A positive hybridization signal was obtained with one sample after three passages in CaCo-2 cells (Figure 3). It has been reported in clinical samples that after CaCo-2 cell passage the minimum number of physical particles required for the specific detection of nucleic acids from infectious rotavirus. astrovirus, andentericadenovirus (Ad401decreasesfrom IO3, IO9.and 1O'to IOz,lO6and IO3,respectively (91. Theaverage efficiency of the virus concentration procedure employed in our studies with water samples has been previously reported to be around 50% (13) Three serial passages in CaCo-2 cells were required to detect astroviruses. The sample containing astroviruses was also positive for enterovirus, rotavirus. and adenovirus 40 and was collected from a dam receiving sewage effluents from a community where a concurrent gastroenteritis outbreak was reponed.
Discussion The discharge of wastewater effluents containing large quantities of animal viruses into surface waters adds a VOL. 29. NO. 10.1995 I ENVIRONMENTAL SCIENCE .%TECHNOLOGY. 2037
considerable amount of viral pollution to the environment (14). Water contaminated with viruses poses a potential human health risk (15). Routine tests for fecal bacterial contamination have been reported to be an inconsistent indication of viral pollution and reports exist of water-borne outbreaks related to water that met bacteriological standards (10, la, supporting the recommendation of direct monitoring of viral pathogens. Current existing virus standards for environmental waters are exclusively based on monitoring the presence of enteroviruses (17). However, several studies conducted on the comparative persistence of entericviruses show that enteroviruses such as poliovirus are not adequate indicators of the behavior of human enteric viruses of major health significance such as rotaviruses or hepatitis A virus (18-20). We describe in these studies a procedure based on the infection of CaCo-2 cells and molecular hybridization with specific cDNA probes for the detection of infectious fastidious enteric viruses in environmental samples. CaCo-2 cells derived from a relatively well-differentiated primary human colon adenocarcinoma (21) form when growing in vitro polarized monolayers with epithelial morphology and well-developedmicrovilli. When observed under the scanning electron microscopy, CaCo-2 cells remember the appearance of actual intestinal cells (data not shown). As a routine, all samples were pretreated with trypsin, since proteolytic enzymes have been described to enhance the infectivity of a wide variety of enteric viruses (22-24). After i n vivo amplification in CaCo-2 cells, enteroviruses, rotaviruses, enteric adenoviruses, and for the first time, astroviruses could be detected in samples of fresh water from a sewage polluted area. Currently, rotaviruses are the most common cause of acute infantile diarrhea worldwide followed by enteric adenoviruses 40 and 41, which account for approximately 10% of acute diarrheal episodes in young children (25). However, some reports presently indicate that astrovirus infections may have been underestimated and are likely to be the second most common cause of viral gastroenteritis in children and adults (26). Following the described methodology, there is now the possibility to monitor the presence of a wide range of actual human viral pathogens in a water source by using a single cell line. Additionally, the procedure enables the specific detection of infectious virus particles, which is of major importance in the evaluation of water treatment and disinfection processes and could be a most valuable tool in the prevention of waterborne viral diseases.
Literature Cied (1) Flores, J.; Boeggeman, E.; Purcell, R. H.; Serena, M.; Perez, I.; White, L.; Wyatt, R. G.; Chanock, R. M.; Kapikian, A. Z. Lancet 1983, 1, 555. (2) Kidd,A. H.; Harley, E. H.; Erasmus, M.J. J. Clin. Microbiol. 1985, 22, 934. (3) Kulski, J. K.; Norval, M. Arch. Virol. 1985, 83, 3. (4) Jehl-Pietri, C.; Hugues, B.; AndrB, M.; Diez, J. M.; Bosch, A. Lett. Appl. Microbiol. 1993, 17, 162. (5) Moore, N. 1.; Margolin, A. B. Appl. Environ. Microbiol. 1993,59, 3145. (6) Eisenstein, B. I. N. Engl. J. Med. 1990, 322, 178. (7) Kopecka, H.; Dubrou, S.; Prevot, J.; Marechal, J.; Lbpez-Pila, J. M. Appl. Environ. Microbiol. 1993, 59, 1213. (8) Tsai, Y.L.; Sobsey, M. D.; Sangermano, L. R.; Palmer, C. J. Appl. Environ. Microbiol. 1993, 59,3488. (9) Pint6, R. M.; Diez, J. M.; Bosch. A. J. Med. Virol. 1994, 22, 310. (10) Bosch, A.; Lucena, F.; Diez, J. M.; Gajardo, R.; Blasi, M.; Jofre, 1.1.Am. Water Works Assoc. 1991, 83, 80. (11) Willcocks, M. M.; Carter, M. J, Arch. Virol. 1992, 124, 279. (12) Racaniello,V.R.; Baltimore, D. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4887. (13) Ma, J.-F.;Naranjo, J.; Gerba, C. P.Appl. Environ. Microbiol. 1994, 60, 1974. (14) Rao, V. C.; Melnick, J. L. Environmental Virology; Cole, A. J., Knowles, C. J., Schlessinger, D., Eds.; Aspects of Microbiology 13; American Society for Microbiology: Washington, DC, 1986. (15) Craun, G. F. J. Environ. Health 1985, 48, 122. (16) Hejkal, T.W.; Keswick, B.; LaBelle, R. L.; Gerba, C. P.; Sanchez, Y.; Dreesman, G.; Hakin, B.; Melnick, J. L. J. Am. Water Works Assoc. 1982, 150, 318. (17) European Community. Directivedu Conseil du 8 DBcembre 1975 concernantlaqualitedeseauxde baignade (76/160ICEE).J.Off: Com. Eur. 1976, 5.02, L 31.1. (18) Sobsey, M. D.;Shields, P. A,; Hauchman, F. S.; Davis, A. L.; Rullman, V.; Bosch, A. In Viral Hepatitis and Liver Disease; Zuckerman, A., Ed.; Alan R. Liss, Inc.: New York, 1988; pp 121124. (19) Abad, F. X.;Pint6, R. M.; Diez, J. M.; Bosch, A. Appl. Environ. Microbiol. 1994, 60, 2377. (20) Abad, F. X.;Pint6 R. M.; Bosch, A. Appl. Environ. Microbiol. 1994, 60,3704. (21) Clarke, J. J. Natl. Cancer Inst. 1977, 58, 209. (22) Almeida,J. D.; Hall, T.; Banatvala, J. E.;Totterdell, B. M.; Chrystie, I. L. J. Gen. Virol. 1978, 40, 213. (23) Lee, T. L.;Kurtz, J. B. J. Gen. Virol. 1981, 57, 421. (24) Bosch, A.; Pint6, R. M.; Blanch, A. R.; Jofre,J. T. WaterRes. 1988, 22, 343. (25) Farthing, M. J. G. In Viruses and the gut; Farthing, M. J. G., Ed.; Smith Kline & French, Ltd.: Welwyn Garden City,Hertfordshire, U.K., 1989; pp 1-4. (26) Willcocks, M. M.; Carter, M. J.; Madeley, C. R. Rev. Med. Virol. 1992, 2, 97.
Received for review February 27, 1995. Revised manuscript received June 8, 1995. Accepted June 16, 1995.@
Acknowledgments We are grateful to the following colleagues for providing virus strains and/or gene probes: T. Cromeans (Centres for Disease Control,Atlanta, GA), W. D. Cubitt (Hospitalfor
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Sick Children, London), J. Flores (National Institutes of Health, Bethesda, MD), C. P. Gerba (University ofArizona, Tucson,AZ),W. 0.K. Grabow (Universityof Pretoria,South Africa), and M. M. Willcocks (University of Surrey).
29, NO. 10,1995
ES950130L @Abstractpublished in Advance ACS Abstracts, August 1, 1995.
