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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples
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Patsy M. Polston a , Roberto A. Rodríguez a,∗ , KyungJin Seo b , Misoon Kim b , GwangPyo Ko b,c , Mark D. Sobsey a a
University of North Carolina at Chapel Hill, Gillings School of Global Public Health, Chapel Hill, NC, USA Seoul National University, Center for Human and Environmental Microbiome, Graduate School of Public Health, Seoul, South Korea c Seoul National University, Bio-MAX Institute, Seoul, South Korea b
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a b s t r a c t
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Article history: Received 7 October 2013 Received in revised form 23 April 2014 Accepted 2 May 2014 Available online xxx
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Keywords: Enteric adenovirus CC/mRNA Detection Real-time PCR Transactivated cell line CMV 293
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1. Introduction
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Human enteric adenoviruses (HAdVs) are commonly detected in waters contaminated with human fecal material and persistent in the environment. Detecting infectious enteric HAdVs is limited by the difficulty of growing them in cell cultures. Recently, an improved cell line (293 CMV) has been described, which enhanced the propagation of enteric HAdVs (Kim et al., 2010. Appl. Environ. Microbiol. 76, 2509). The present study evaluated the transactivated 293 CMV cell line for detecting enteric HAdVs from field samples, which is an important step in demonstrating the usefulness of the improved cell line for water monitoring programs. Field samples consisted of the following: concentrated sewage samples (from 1 L) collected from three different wastewater treatment plants (WWTPs) and concentrated raw source water samples (from 20 L) collected from six water treatment plants (WTPs). Infectious HAdVs were detected using a combined cell culture/mRNA RT-PCR assay. Concentrated samples were assayed, in parallel, using the standard (STD) G293 and 293 CMV cell lines. Viral replication was determined by measuring viral mRNA and viral DNA levels during infection. Infectious HAdVs were successfully detected from environmental samples using the new transactivated and standard cell lines. Infectivity assays of concentrated sewage samples demonstrated higher viral mRNA expression (p = 0.02) and viral DNA concentrations (p = 0.02) in the transactivated 293 CMV than in the G293 cell line. Although not statistically significant, infectious HAdVs were detected in more raw water samples using the 293 CMV cells (8 of 18) than in the STD G293 cells (4 of 18). However, when results of the source water samples were pooled, the number of flasks positive using the 293 CMV cells was significantly greater than those using the G293 cells (p = 0.01). Overall, the results of the present study demonstrate the effectiveness of the new transactivated 293 CMV cell line for improved propagation and detection of HAdVs from environmental samples. © 2014 Published by Elsevier B.V.
Human adenoviruses (HAdVs) are non-enveloped, linear dsDNA viruses of the Adenoviridae family. Adenoviruses are widely present in ambient waters, persist over long periods of time in the water environment (Enriquez et al., 1995; He and Jiang, 2005), and are extremely resistant to inactivation by UV (Ko et al., 2005; Thurston-Enriquez et al., 2003). HAdV abundance and persistence presents challenges for disinfection by water treatment plants (WTPs) and they have been detected consistently in disinfected
∗ Corresponding author at: School of Public Health—EL Paso Regional Campus, University of Texas Health Science Center at Houston, 1101 N. Campbell CH414, EL Paso, TX 79902, USA. Tel.: +1 915 747 8538. E-mail address: [email protected] (R.A. Rodríguez).
sewage (Rodriguez et al., 2008; Sedmak et al., 2005; Simmons et al., 2011). HAdVs have also been detected frequently in drinking water, surface water, groundwater, and recreational waters (Aslan et al., 2011; Bofill-Mas et al., 2010; Keswick et al., 1984; van Heerden et al., 2005; Wyn-Jones et al., 2011). After rotaviruses, enteric HAdV (40 and 41) are the leading causes of childhood diarrhea and most abundant HAdV species detected in surface waters and sewage (Mena and Gerba, 2009). For the above reasons, HAdVs are one of the microbiological agents included in the United States Environmental Protection Agency’s drinking water contaminant candidate list (CCL) (Unites States Environmental Protection Agency, 2010). Considering the public health impact and ongoing concern, it is important to develop methods that detect and quantify infectious HAdVs found in environmental samples, such as sewage and water, which pose health risks from waterborne exposures.
http://dx.doi.org/10.1016/j.jviromet.2014.05.002 0166-0934/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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The detection of HAdVs from environmental samples is usually performed by PCR or cell culture methods (Jiang, 2006). Although direct PCR is a rapid and robust approach for virus detection, the 51 infectivity of detected viruses is uncertain and difficult to establish 52 from PCR results (Richards, 1999). Detecting enteric adenoviruses 53 by cell culture infectivity is usually problematic due to the incon54 55Q3 sistent onset of viral induced cytopathic effect (CPE). Furthermore, detection using the PLC/PRF/5 cell line could take 10 to 20 days 56 and several cell passages may be required before viral presence 57 becomes apparent (Thurston-Enriquez et al., 2003). The combined 58 use of cell culture with PCR has been used for detecting adenovi59 rus. This combined method can be used for detecting the presence 60 of viral genome (CC–PCR) or for detecting viral mRNA (CC Reverse 61 transcription PCR, Ko et al., 2003). Recently, a stable cell line was 62 developed to promote the propagation of enteric HAdVs. This cell 63 line consistently expressed high levels of viral transactivating IE1 64 protein of cytomegalovirus (CMV) (Kim et al., 2010). The CMV viral 65 transactivated protein can activate and stimulate viral genes and 66 transcription factors from enteric HAdVs, which aid to increase viral 67 mRNA levels and promote propagation of these fastidious human 68 adenoviruses in cell culture (Kim et al., 2010). This cell line has only 69 been tested with laboratory reference strains and clinical isolates, 70 and its utility in detecting HAdVs from environmental samples has 71 not yet been determined. 72 The goal of this study was to test the feasibility of using the 73 improved cell line (293 CMV) for detecting enteric HAdVs, using 74 environmental sewage and water samples collected from differ75 ent surface water and wastewater sources. Detection of enteric 76 adenovirus was compared between the standard (G293) and the 77 improved transactivated (293 CMV) cell lines. For this study, the 78 replication of HAdV in the cell line was determined by mea79 suring the production of viral mRNA and determining the levels 80 of viral DNA; therefore, the results are representative of what 81 could be expected when using this improved cell line for either 82 a cell culture–qPCR or a cell culture mRNA RT qPCR approach. To 83 our knowledge, this is the first report using this improved cell 84 line for detecting infectious adenovirus from environmental water 85 samples. 86 49
2.3. Source water sample collection and concentration
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2. Materials and methods 2.1. Adenovirus type 41 stock The adenovirus type 41 stock (ATCC, VR-930) was propagated using the G293 host cells in 150 cm2 tissue culture flasks and purified by PEG as described by Rodríguez et al. (2013). The viral titer was determined using the mRNA qRT-PCR method described by Rodríguez et al. (2013).
2.2. Sewage samples Sewage samples were collected from three wastewater treatment plants (WWTP) in North Carolina. One liter was collected and concentrated, using organic flocculation, and polyethylene glycol precipitation (PEG) to a final volume of 5 mL as described by Rodríguez et al. (2013). To kill any bacteria and remove soluble impurities, 5 mL of chloroform was added to the sample and mixed to emulsify by inverting the tubes vertically for one minute. The samples were centrifuged (3000 × g, 20 min, 4 ◦ C). The supernatant was recovered, without disturbing the thin film layer above the chloroform, yielding a final sample volume of 5 mL. The supernatant was stored at −80 ◦ C and later assayed for viruses.
Samples were collected from six geographically distinct water treatment plants, within the United States, during three different sample times (September 2009, November 2009, and February 2010) per plant, yielding 18 samples. Each facility collected 24 L of raw source water. Samples were stored overnight at 4 ◦ C before shipping, by next day air express, to our laboratory. The temperature of the samples was recorded using a data logger system and never surpassed 7 ◦ C. Upon arrival at the UNC laboratory, 20 L samples were concentrated using hollow fiber ultrafiltration (HFUF), polyethylene glycol precipitation (PEG), and ultracentrifugation procedures in succession to a final volume of approximately 5 mL. The concentrated samples were stored in −80 ◦ C for future cell culture analysis. Twenty-liter volumes of source water were initially concentrated using tangential flow HFUF with a Hemoflow F80A sterile ultra-filter cartridge (molecular weight cut-off 15,000 to 20,000). The source water was re-circulated using a peristaltic pump at pressure between 15–25 psi until the recirculating water volume was emptied in the cubic polyethylene container holding the initial sample. To recover any viruses attached to the filter, the viruses were eluted by adding 250 mL of HFUF eluting solution (1XPBS/1% Laureth-12) consisting of 10 g/L of Laureth-12 and 50 L antifoam-A per 1 L of PBS (pH 7.4). The resulting volume of retentate was approximately 200–300 mL. The retentate was concentrated further using PEG precipitation at a final concentration of 9% of PEG 8000 (Fisher, Cat no. 233-1, or equivalent) and 0.3 M NaCl; the sample was separated into 150 mL volumes using 250 mL conical centrifuge bottles. The bottles were placed on an orbital shaker and allowed to shake overnight (200 rpm, 4 ◦ C). The next day, the bottles were centrifuged (5400 × g, 1 h, 4 ◦ C). The supernatant was decanted and pellets were resuspended using 15 mL of PBS (pH 7.5). The resulting water sample concentrates were combined and stored in a 50 mL polypropylene centrifuge tube. To kill any bacteria and to remove certain soluble impurities, 5 mL of chloroform was added to the concentrated sample from step 2 (PEG precipitation) and mixed to emulsify by inverting the tubes in vertical movement for one minute. The samples were centrifuged (3000 × g, 20 min, 4 ◦ C), the supernatant was recovered from the top without disturbing the thin film layer above the chloroform, and the volumes were recorded. The supernatant was stored at −80 ◦ C or ultracentrifugation immediately followed. The volume of the supernatant recovered after chloroform extraction (in the previous step) was adjusted to 50 mL by adding PBS (pH 7.5). The sample was transferred to a 75 mL ultracentrifuge tube and centrifuged (100,000 × g, 4 h, 4 ◦ C). Ultracentrifugation separated the viruses from the buffer and further reduced the sample volume to 5 mL. The supernatant was immediately decanted and the pellet was resuspended in 5 mL of PBS (pH 7.5). The concentrated samples were aliquoted in four 1.2 mL tubes and stored at −80 ◦ C for subsequent virus infectivity assay. 2.4. Cell culture infections and detection of adenovirus mRNA and DNA during cell culture infectivity assays The infectivity assays were performed as described by Rodríguez et al. (2013). A 1.5 mL inoculum was produced by diluting 350 L of concentrated sample with 1050 L complete MEM medium, without serum and containing 10 g kanamicin, 50 g gentamicin, and 20 g nystatin per mL of medium. The use of media in the inocula reduced cell detachment from their substrate after inoculation. After one hour of incubation at 37 ◦ C for viral adsorption, the inoculum was removed, 6 mL of complete MEM medium with 2% fetal bovine serum was added to each flask, and the cell cultures were incubated at 37 ◦ C in a water-jacked CO2 incubator.
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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2.5. Detection of adenovirus mRNA and DNA during cell culture infectivity assays
Detection of adenovirus mRNA in cell culture infectivity assays 173 was performed as described by Rodríguez et al. (2013). The nucleic 174 acids from the cell monolayers inoculated with concentrated water 175 samples were extracted and purified, as described by the manu176 facturer of the Qiagen RNeasy kit (Qiagen, Valencia, CA). The final 177 nucleic acid extract was split into two 25 L volumes. One volume 178 was treated with DNAse (Promega, Madison, WI) to remove the 179 DNA from the sample and used for mRNA analysis. The second 180 untreated aliquot was used for quantifying the adenovirus DNA. 181 The reverse transcription step was performed using the M-MLV 182 reverse transcriptase (Invitrogen, Carlsbad, CA) and the oligo dT18 183 primer, as described previously (Rodríguez et al., 2013). The cel184 lular housekeeping gene, GAPDH, was used as an internal control 185 for the mRNA assay. Real-time PCR for the quantification of cDNA 186 and viral DNA used the primer-probe set specific for the hexon 187 gene of human adenovirus (species A to F) as published by Jothiku188 Q4 mar et al. (2005). The real-time PCR assay was performed using 189 the QuantiTeck probe PCR master mix (Qiagen, Valencia, CA). The 190 concentration of the primers and dual-labeled probe were 1.0 M 191 and 0.1 M, respectively. The real-time PCR was run in a SmartCy192 cler (Cepheid, Sunnydale, CA) and the cycling conditions were as 193 follows: initial denaturation for 15 min at 95 ◦ C and then 45 cycles 194 of the following: 15 s at 95 ◦ C, 30 s at 58 ◦ C, and 15 s at 72 ◦ C. The 195 control used for calibration was created by inserting a fragment of 196 the adenovirus hexon gene into a TOPO clone as described previ197 ously (Rodríguez et al., 2012). The PCR efficiency was 110% and the 198 calculated detection limit was 1.5 genome copies per PCR reaction 199 (2 L assayed). Ct values were transformed into log10 copies/PCR 200 reaction and then the concentration was adjusted per 50 L of final 201 volume of nucleic acid obtained after the purification of the viral 202 contents of a 25 cm2 flask. 172
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2.6. Comparison of transactivated 293 CMV and standard G293 cell lines for the detection of HAdV 41 and HAdV from sewage and source water samples To demonstrate the performance of the transactivated cell line to promote the growth of enteric HAdVs, two different HAdV 41 concentrations were used, 50 and 5 infectious units, respectively. All the assays were performed in parallel and the propagation of HAdVs was confirmed using real-time RT-PCR targeting the adenovirus hexon gene. The concentrations of HAdVs in sewage samples were determined using the combined cell culture and real time RT-PCR assay described previously for detecting adenoviral mRNA (Rodríguez et al., 2013). Comparison between cell lines for adenovirus detection by infectivity assays were performed, in parallel, using a purified concentrated sewage sample as inoculum and adjusting to adenovirus concentrations of 10 infectious units (IU), 2 IU, and 0.8 IU per well. Adenovirus propagation was determined by measuring viral mRNA and viral DNA at 2 days post infection (DPI), 3DPI, and 5DPI as described later in the manuscript. To compare the growth of different concentrations of HAdV-41 stock and sewage samples, 3 cell culture flasks per sample were used. Detection of HAdV from surface water by each cell line was conducted for 18 surface water samples as described before. Concentrated sample aliquots of 350 L were mixed with 1 mL of MEM with no BSA, and this mixture was used as inoculum for cell culture infection. Five 25 cm2 flasks containing the cell monolayer were used per sample. In the case of standard G293 cell line, six flasks per sample were used with one flask used to test cell toxicity of the sample inoculum. For the 293 CMV cell line, the flasks were incubated for three days and for the G293 cell line, the flask were incubated for five days. The different incubation times corresponded to optimal
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times required for viral nucleic acid detection as established previously. Adenoviruses from positive flasks were typed by sequencing and amplifying a portion of the hexon gene using the primers hex1 and hex2 described by Ko et al. (2003). 2.7. Statistical analysis An analysis of variance (ANOVA) was used to compare the expression of viral mRNA and concentration of viral DNA during cell culture propagation in the two cell lines (G293 and 293 CMV). For this analysis, the concentrations were transformed into log10 units. Chi square analysis was used to compare the frequency of detection of infectious HAdVs from source water samples between the two cell lines, G293 and 293 CMV. For this comparison, the detection of viral mRNA or viral DNA was used as indication of infection, and the results were expressed as flasks either positive or negative for viral replication. Each concentrated source water sample was analyzed with each cell line in parallel, as previously described. All statistical analyses were performed using the Minitab program (release 14.23, Minitab Inc., State College, PA). 2.8. Quality assurance and control Samples of source water were cooled at 4 ◦ C and shipped overnight in coolers along with a chain of custody form, which was kept for each sample. The sample sheet, custody form, supporting information, and results were archived. The water samples (from WTPs) and sewage samples (from WWTPs) received in NC were processed and analyzed in a lab room dedicated specifically for field samples. The room was used exclusively for processing field samples and no work with positive controls, microbial reference strains, or their nucleic acids was performed in that room. Nucleic acid extractions and assays of samples in cell culture were also performed within the field samples room. Positive controls were processed in a separate lab room, which was dedicated for working with known pathogens and involved the use of positive control and reference strains. The different stages in the analysis of water samples was done on a schedule such that all samples being processed were at the same stage of analysis and no work at different stages of analysis was performed simultaneously. The analysis of PCR product was the last step and was conducted in a separate lab room designated for PCR amplifications and related PCR product analysis. All equipment and reusable labware was autoclaved or decontaminated with sodium hypochlorite, depending on the nature of the equipment and labware. 3. Results 3.1. Comparison of the performance of the new transactivated 293 CMV cell line with the standard G293 cell line for the propagation of adenovirus 41 The propagation of HAdV 41 was compared between the G293 and 293 CMV cell lines. The expression of the viral hexon gene was consistently higher in the 293 CMV cell line than in the G293 cell line, but the magnitude of difference declined with longer incubation time (Fig. 1). The difference observed in mRNA levels per cell line was statistically significant (p = 0.01). 3.2. Comparison of the transactivated 293 CMV cell line with the standard G293 cell line for the detection of human adenovirus in concentrated sewage samples The propagation of HAdV from the collected sewage in the new transactivated 293 CMV cell line at very low concentrations was
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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the incubation time is longer, the time to maximum detection by the 293 CMV cell line is earlier. The results for the WWTP-3 samples yielded mostly non-detectable values; therefore, this sample was not used for quantitative comparison. Positives flasks were analyzed by sequencing and most of the adenoviruses detected were from subgroup F. Adenoviruses subgroup A were detected but in only 2 flasks. No apparent differences were observed in which adenovirus types were detected between the improved and standard cell lines.
3.3. Comparison of the G293 cell line with the 293 CMV cell line for the detection of infectious human adenovirus in source water sample concentrates
Fig. 1. Comparison of transactivated 293 CMV (䊉) cells with G293 cells () for the propagation of adenovirus 41 as measured by the production of viral hexon gene mRNA at initial viral inocula of 5 infectious unit (100 genome copies) per flask and 50 infectious units (1000 genome copies) per flask. The titer of the stock was determined both by direct PCR and by infectivity assay. Reported concentrations are the average of three experiments (n = 3) and the units are log10 copies per well; error bars represent standard deviation.
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demonstrated, as shown in Table 1. Significantly higher concentrations of viral mRNA (p = 0.015) and viral DNA (p = 0.045) were observed in the 293 CMV monolayers than in the G293 monolayers. WWTP-1 samples demonstrated greater numbers of positive samples detected compared to other sample sources, when targeting viral hexon gene mRNA and viral hexon gene DNA for both cell lines. The infectivity assays conducted for HAdVs in WWTP-2 yielded detectable levels of both viral mRNA and viral DNA in the new transactivated 293 CMV cell line as early as 2 DPI. For the standard G293 cells, HAdVs were detected only at 5 DPI. Notably, the viral DNA level detected for G293, which was the only day with detectable results, falls within the range of detectable values for 293 CMV cells. Although the G293 cell line eventually reaches similar levels of adenovirus hexon gene mRNA detection as in the 293 CMV cell line if
Source water samples from different locations in the US were concentrated by the successive steps of HFUF, PEG precipitation, and ultracentrifugation and used to compare human adenovirus detection in the transactivated 293 CMV and G293 cell lines. Initial testing was conducted with concentrated WTP source water samples to determine possible interferences from sample cytotoxicity. Concentrated sample 2 from WTP-E showed high toxicity that manifested as cell death; therefore, this sample was further diluted two-fold with maintenance medium (no serum) prior to performing the infectivity assays as this dilution reduced the toxicity of the sample. All other concentrated samples did not show toxicity when inoculated at 350 L concentrated sample volumes per cell culture. All WTP source waters were subsequently analyzed using the 293 CMV cell line (Table 2). The inoculated cells were incubated for 3 days as previous results showed the highest expression of viral hexon gene nucleic acid was observed at this incubation time for 293 CMV cells. The equivalent volume of source water analyzed for each sample was 5 L, except for the second sample collected from WTP-E, for which the sample volume was reduced to 1.6 L of source water per sample in order to reduce cell toxicity. Overall, 8 source water samples were positive for infectious adenoviruses hexon gene nucleic acid using the transactivated 293 CMV cell line, and 4 samples were positive using the G293 cell line, of all 18 source water samples collected and analyzed, as shown in Table 2. Three samples were positive with both cell lines, while
Table 1 Comparison of 293 CMV and G293 cell lines for the propagation of human adenoviruses in sewage samples from three different waste water treatment plants (WWTPs). Sewage sample
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Initial adenovirus concentration Infectious units (IU)a
Genome copies (GC)b
11
240
G293 cell line
CMV
STD
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2
44
CMV
STD
3
0.8
11
CMV STD
DPIc
2 3 5 2 3 5 2 3 5 2 3 5 3 5 3 5
Adenovirus replication outcome Viral mRNA (total 3 wells)
Viral DNA (total 3 wells)
Positive
Log10 GC (±STD)
Positive
Log10 GC (±STD)
3 3 3 3 3 3 2 3 2 0 0 2 0 1 0 0
3.1±0.1 3.5±0.3 2.6±0.2 3.0±0.0 2.4±0.6 1.9±0.9 1.7 2.5±0.8 1.6 NDd ND 1.6 ND 0.7 ND ND
3 3 3 3 3 3 2 3 3 0 0 3 0 2 1 2
3.4±0.1 4.1±0.3 4.7±0.4 2.5±0.8 3.5±0.8 4.5±0.0 1.9 3.2±0.7 3.6±0.7 ND ND 3.5±0.6 ND 2.2 1.3 1.6
a Infectious units per flask used in the inoculum were determined using mRNA reverse transcription PCR in infected G293 cell lines as described previously (Rodríguez et al., 2013). b Genome copies per flask used in the inoculum were determined using real-time PCR as described previously (Rodríguez et al., 2013). c Days post infection. d None detected after 45 cycles.
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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Table 2 Comparison of results for human adenovirus infectivity assays of source water samples between the standard (G293) and improved (293 CMV) cell lines. Sitea
A
B
C
D
E
F
293 CMV
Sampleb
1 12 18 2 11 17 3 7 15 4 9 16 6 8 13 5 10 14
Total
G293
mRNAc (positive/total)
DNAd (positive/total)
Combined resultse
mRNA (positive/total)
DNA (positive/total)
Combined results
2/5 0/5 0/5 0/5 1/5 0/5 0/5 2/5 2/5 1/5 2/5 0/5 0/5 1/5 1/5 0/5 0/5 0/5 13/90
1/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 1/5 2/5 0/5 0/5 1/5 1/5 0/5 0/5 0/5 8/90
Positive Negative Negative Negative Positive Negative Negative Positive Positive Positive Positive Negative Negative Positive Positive Negative Negative Negative 8/18
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 1/6 0/6 2/6 0/6 0/6 0/6 1/6 0/6 0/6 0/6 4/108
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 1/6 0/6 0/6 1/6 0/6 0/6 2/6 0/6 0/6 0/6 4/108
Negative Negative Negative Negative Negative Negative Negative Negative Positive Negative Positive Positive Negative Negative Positive Negative Negative Negative 4/18
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Source water sample collection site (water treatment plant). Twenty liter source water sample. The sample numbers are in order of collection. Samples 1 to 6 were collected in September 2009, samples 7 to 12 were collected in December 2009, and samples 13–18 were collected in March 2010. c Detection of adenovirus mRNA in cell monolayers after cell culture infection. The analysis times for 293 CMV cell lines were performed after 3 days post-incubation and for G293 cell line after 5 days post-incubation. d Detection of adenovirus DNA in cell monolayers after cell culture inoculation and incubation. Time of analysis for viral DNA was the same as for mRNA. e Cell culture result per sample, based positivity for DNA or mRNA scored as positive. b
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1 sample was positive when analyzed with the G293 cell line and negative with the 293 CMV cell line. Five samples were positive when assayed with the 293 CMV cell line and negative when analyzed with the G293 cell line. More samples were positive within the 293 CMV cell line as compared to the G293 cell line; however, the increase in samples positive for virus detection was not significant as determined by chi square analysis (p > 0.1). For this analysis, a sample was considered positive when one of the flasks was positive by detecting either the viral mRNA or viral DNA. However, the number of cell culture flasks positive for HAdV as determined by presence of either viral mRNA or viral DNA, was significant with more flasks positive for the 293 CMV cell line (21 of 90) than the G293 cell line (8 of 108) (Table 2, p = 0.002).
4. Discussion The evaluation of the new transactivated 293 CMV cell line for the detection of wild type adenoviruses viruses at concentrations encountered in environmental samples is important evidence for demonstrating the utility of the improved cell line in monitoring the presence of adenovirus in the water environment. This study demonstrated the effectiveness of this new cell line for the detection of infectious HAdV from sewage and source water samples. The use of an improved cell line resulted in an earlier increase in viral replication when assaying concentrated sewage samples and more positive source water samples at shorter incubation times. The transactivated 293 CMV cell line was created from the parent G293 cell line, which is a cell line containing a portion of the HAdV 5 genome that is used commonly for the propagation of enteric adenovirus. This initial genetic modification resulted in a cell line capable of supporting efficient growth of enteric HAdV (Graham et al., 1977). However, enteric HAdV shows a variable and inconsistent manifestation of viral cytopathogenic effects in the G293 cell line. The inconsistent onset of CPE of enteric HAdV in cell culture is mostly due to the poor expression of early genes, the inability to evade the cell immune response, and the delay in the release of the mature viral particles (Brown et al., 1992; Mautner et al.,
1999). For example, interferon deficient cells created by expressing the V protein of paramyxovirus simian virus 5 resulted in better propagation of enteric HAdV (Sherwood et al., 2007). The 293 CMV cell line contains the gene to produce transactivated protein IE1 from cytoglomegavirus that promotes the expression of HAdV early genes, resulting in higher levels of viral mRNA and viral DNA when compared to the G293 cell line (Kim et al., 2010). As demonstrated in the present study, there was increased earlier expression of HAdV mRNA at higher levels in the 293 CMV cell line than in the G293 cell line, resulting in faster detection of HAdV isolates from environmental samples. The assessment of the transactivated cell line in this work was designed to demonstrate the early detection of human adenovirus at very low concentrations, particularly of environmental strains (wild types), determine the time required for detection of environmental adenovirus and document the ability of the transactivated cell line to monitor the presence of infectious HAdVs in environmental samples. Sewage samples were used to determine the detection and propagation of adenovirus at concentrations that are relevant to analysis of environmental samples. A sewage sample (number 2) showing no HAdV detection in the first two incubation times for the standard cell line resulted in HAdV detection as early as 2 days for the improved cell line (Table 1). Because infectivity assays in both cell lines were performed in parallel using the same inoculum and internal control, these results provide evidence that the improved cell line is capable of earlier detection of human adenovirus. However results observed in another sewage sample (number 3) were less conclusive because the concentrations of adenovirus in those assays were almost below the detection limit of one infectious unit per flask and therefore would require more replicate assays to detect differences in HAdV detection by the two cell lines (Rodríguez et al., 2013). Improving infectivity assays for enteric adenoviruses is challenging because environmental isolates tend to have different growth kinetics than corresponding laboratory-adapted isolates. For example, during the development of a plaque assay for enteric adenovirus, multiple isolates were tested and selected for their capacity to produce CPE (Cromeans et al., 2008). After selection and Q5
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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several passages in cell cultures isolates producing stable plaques were obtained, making this assay specific for the selected enteric adenovirus strain. This assay for the cell-culture adapted HAdV has been useful for studying the stability of the virus in the environment (Rigotto et al., 2011) and could be useful for determining disinfection performance and the performance of recovery methods. However, because this plaque assay is isolate-specific, its applicability to the detection of wild type infectious adenovirus found in the environment may be limited. Other improved cell culture assays for adenovirus are based on detecting viral proteins during cell culture propagation using the 293 cell line and flow cytometry. This approach has been able to quantify human adenovirus from environmental samples (Li et al., 2010). Initial assessment of the transactivated cell line 293 CMV was performed using laboratory adapted strains of enteric adenovirus and clinical strains obtained from stool samples (Kim et al., 2010). The use of a clinical strain of adenovirus 40 helped demonstrate the applicability of the transactivated cell lines to non-cell culture adapted viral isolates, but this initial assessment was performed using higher viral titers not relevant to the much lower viral titers of most environmental samples (Kim et al., 2010). A new and improved 293 cell line (293 RAS) that overexpresses the RAS protein has also been developed that has demonstrated enhanced capability to propagate enteric adenovirus at low concentrations (Si et al., 2013). Comparison experiments between the 293 CMV and the 293 RAS cell lines to detect wildtype human adenoviruses from environmental samples are now warranted. HAdV has been proposed for monitoring human fecal contamination in water because of its continuous presence in sewage samples and its detection in fecally contaminated waters (Silva et al., 2011). Another advantage of the detection of HAdV in sewage and sewage-contaminated waters is its strict human origin, as it has not been detected in any other host besides humans (Ahmed et al., 2010)., Despite previous attempts to use different cell culture approaches (Jiang, 2006), determining HAdV infectivity in environmental samples is challenging. The detection of HAdV infectivity in environmental samples using the onset and appearance of cytopathic effects in the cell monolayer has been reported previously (Jiang et al., 2009). However, the detection of viral replication intermediates, such as the approach demonstrated in this study for early detection of viral mRNA, can result in faster detection because it is not necessary to wait for the onset of viral induced cytopathic effect (Ko et al., 2003). In addition to a previous study on detecting HAdV in environmental samples by combined cell culture, qPCR and mRNA RT-qPCR in 293 cells (Rodríguez et al., 2013), Ogorzaly et al. (2013) have also reported the detection of HAdV DNA by qPCR in environmental samples using a sub-clone of 293 cells, called 293A. It should be noted, their study design focused on only DNA to detect HAdV and not RNA, as done in the protocol. Their results for detecting HAdV DNA are similar to those described above, in that detection of HAdV DNA was possible within 2 days and did not increase further by 3 days. However, it is not possible know if the cell culture infectivity protocol and 293 cell line used in their study and in the current study are comparable or differ in performance unless compared in parallel for the same viral targets of mRNA and DNA on the same environmental samples simultaneously, as done in comparing standard 293 cells to 293 CMV cells. Such further comparisons are recommended. Similarly, the appearance of viral DNA (Chapron et al., 2000; Dong et al., 2010) or viral early proteins (Calgua et al., 2011; Li et al., 2010) also have been targets for more rapid detection of HAdVs than the visual appearance of CPE in other studies. The improved transactivated 293 CMV cell line could be adapted to any of these approaches for infectivity detection of early viral replication products, possibly improving their performance and decreasing detection time.
In the present study, detecting infectious HAdV by detecting the viral mRNA using the 293 CMV cell line resulted in better detection (greater numbers of positive cell cultures) and shorter incubation times than possible with the standard G293 cell line. In addition, as demonstrated by Kim et al. (2010), onset of cytopathic effect on the 293 CMV cell line is clearer than in the standard G293 cell line. Therefore, the 293 CMV cell line may better facilitate the development of plaque assays that detect infectious enteric HAdV in environmental samples. In the analysis of source water samples, greater numbers of flasks were positive by analyzing the production of adenovirus mRNA during cell culture than analyzing for the viral DNA. This may be because more copies of mRNA per DNA are produced during early stages of viral replication in cell culture infectivity assays (Ko et al., 2003). However, as infection progresses, mRNA levels remain constant and DNA levels increase, as was observed in the results of Table 1. Therefore, longer incubation time could have resulted in the detection of viral DNA in samples where only mRNA was detected initially. Although the nucleic extraction method used in this experiment was optimized for extraction of mRNA, detection of viral DNA in the experiments with sewage samples was consistent with the level of inoculum and incubation time, demonstrating that this method also was efficient for the recovery of viral DNA. In conclusion, the results of this study indicate that the new transactivated 293 CMV cell line demonstrated improved ability to rapidly detect low levels of infectious human adenoviruses from sewage and environmental water samples than the standard G293 cell line. In addition, the use of transactivated 293 CMV cells resulted in faster replication of human adenoviruses found in environmental sewage and water samples when compared to the G293 cells. This conclusion is based on earlier detection of greater amounts of viral hexon gene mRNA by a sensitive and specific quantitative RT-PCR assay applied to nucleic acid extracts of infected cell cultures. The results of this study also provide evidence that the new 293 CMV cell line may detect more infectious human adenoviruses than did the standard G293 cell line in environmental source water samples. Use of this new 293 CMV cell line has the potential to provide more rapid and potentially increased detection of infectious human viruses in environmental water and wastewater samples; thereby, improving monitoring and surveillance of infectious human enteric adenoviruses in these environmental waters for more timely management decisions and response actions. Acknowledgments This work was funded by the Water Research Foundation Q6 (Project Number 3181) and the Agriculture Research Center pro- Q7 gram of the Ministry for Food, Agriculture, Forestry and Fisheries, Korea. We would like to thank to the facility managers of the water and wastewater treatment plants for agreeing to participate in this project and providing environmental samples. References Ahmed, W., Goonetilleke, A., Gardner, T., 2010. Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia. Water Res. 44, 4662–4673. Aslan, A., Xagoraraki, I., Simmons, F.J., Rose, J.B., Dorevitch, S., 2011. Occurrence of adenovirus and other enteric viruses in limited-contact freshwater recreational areas and bathing waters. J. Appl. Microbiol. 111, 1250–1261. Bofill-Mas, S., Calgua, B., Clemente-Casares, P., La Rosa, G., Iaconelli, M., Muscillo, M., Rutjes, S., Husman, A.M., Grunert, A., Graeber, I., Verani, M., Carducci, A., Calvo, M., Wyn-Jones, P., Girones, R., 2010. Quantification of human adenoviruses in European recreational waters. Food Environ. Virol. 2, 101–109. Brown, M., Wilson-Friesen, H.L., Doane, F., 1992. A block in release of progeny virus and a high particle-to-infectious unit ratio contribute to poor growth of enteric adenovirus types 40 and 41 in cell culture. J. Virol. 66, 3198–3205.
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples
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Patsy M. Polston a , Roberto A. Rodríguez a,∗ , KyungJin Seo b , Misoon Kim b , GwangPyo Ko b,c , Mark D. Sobsey a a
University of North Carolina at Chapel Hill, Gillings School of Global Public Health, Chapel Hill, NC, USA Seoul National University, Center for Human and Environmental Microbiome, Graduate School of Public Health, Seoul, South Korea c Seoul National University, Bio-MAX Institute, Seoul, South Korea b
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Article history: Received 7 October 2013 Received in revised form 23 April 2014 Accepted 2 May 2014 Available online xxx
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Keywords: Enteric adenovirus CC/mRNA Detection Real-time PCR Transactivated cell line CMV 293
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1. Introduction
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Human enteric adenoviruses (HAdVs) are commonly detected in waters contaminated with human fecal material and persistent in the environment. Detecting infectious enteric HAdVs is limited by the difficulty of growing them in cell cultures. Recently, an improved cell line (293 CMV) has been described, which enhanced the propagation of enteric HAdVs (Kim et al., 2010. Appl. Environ. Microbiol. 76, 2509). The present study evaluated the transactivated 293 CMV cell line for detecting enteric HAdVs from field samples, which is an important step in demonstrating the usefulness of the improved cell line for water monitoring programs. Field samples consisted of the following: concentrated sewage samples (from 1 L) collected from three different wastewater treatment plants (WWTPs) and concentrated raw source water samples (from 20 L) collected from six water treatment plants (WTPs). Infectious HAdVs were detected using a combined cell culture/mRNA RT-PCR assay. Concentrated samples were assayed, in parallel, using the standard (STD) G293 and 293 CMV cell lines. Viral replication was determined by measuring viral mRNA and viral DNA levels during infection. Infectious HAdVs were successfully detected from environmental samples using the new transactivated and standard cell lines. Infectivity assays of concentrated sewage samples demonstrated higher viral mRNA expression (p = 0.02) and viral DNA concentrations (p = 0.02) in the transactivated 293 CMV than in the G293 cell line. Although not statistically significant, infectious HAdVs were detected in more raw water samples using the 293 CMV cells (8 of 18) than in the STD G293 cells (4 of 18). However, when results of the source water samples were pooled, the number of flasks positive using the 293 CMV cells was significantly greater than those using the G293 cells (p = 0.01). Overall, the results of the present study demonstrate the effectiveness of the new transactivated 293 CMV cell line for improved propagation and detection of HAdVs from environmental samples. © 2014 Published by Elsevier B.V.
Human adenoviruses (HAdVs) are non-enveloped, linear dsDNA viruses of the Adenoviridae family. Adenoviruses are widely present in ambient waters, persist over long periods of time in the water environment (Enriquez et al., 1995; He and Jiang, 2005), and are extremely resistant to inactivation by UV (Ko et al., 2005; Thurston-Enriquez et al., 2003). HAdV abundance and persistence presents challenges for disinfection by water treatment plants (WTPs) and they have been detected consistently in disinfected
∗ Corresponding author at: School of Public Health—EL Paso Regional Campus, University of Texas Health Science Center at Houston, 1101 N. Campbell CH414, EL Paso, TX 79902, USA. Tel.: +1 915 747 8538. E-mail address: [email protected] (R.A. Rodríguez).
sewage (Rodriguez et al., 2008; Sedmak et al., 2005; Simmons et al., 2011). HAdVs have also been detected frequently in drinking water, surface water, groundwater, and recreational waters (Aslan et al., 2011; Bofill-Mas et al., 2010; Keswick et al., 1984; van Heerden et al., 2005; Wyn-Jones et al., 2011). After rotaviruses, enteric HAdV (40 and 41) are the leading causes of childhood diarrhea and most abundant HAdV species detected in surface waters and sewage (Mena and Gerba, 2009). For the above reasons, HAdVs are one of the microbiological agents included in the United States Environmental Protection Agency’s drinking water contaminant candidate list (CCL) (Unites States Environmental Protection Agency, 2010). Considering the public health impact and ongoing concern, it is important to develop methods that detect and quantify infectious HAdVs found in environmental samples, such as sewage and water, which pose health risks from waterborne exposures.
http://dx.doi.org/10.1016/j.jviromet.2014.05.002 0166-0934/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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The detection of HAdVs from environmental samples is usually performed by PCR or cell culture methods (Jiang, 2006). Although direct PCR is a rapid and robust approach for virus detection, the 51 infectivity of detected viruses is uncertain and difficult to establish 52 from PCR results (Richards, 1999). Detecting enteric adenoviruses 53 by cell culture infectivity is usually problematic due to the incon54 55Q3 sistent onset of viral induced cytopathic effect (CPE). Furthermore, detection using the PLC/PRF/5 cell line could take 10 to 20 days 56 and several cell passages may be required before viral presence 57 becomes apparent (Thurston-Enriquez et al., 2003). The combined 58 use of cell culture with PCR has been used for detecting adenovi59 rus. This combined method can be used for detecting the presence 60 of viral genome (CC–PCR) or for detecting viral mRNA (CC Reverse 61 transcription PCR, Ko et al., 2003). Recently, a stable cell line was 62 developed to promote the propagation of enteric HAdVs. This cell 63 line consistently expressed high levels of viral transactivating IE1 64 protein of cytomegalovirus (CMV) (Kim et al., 2010). The CMV viral 65 transactivated protein can activate and stimulate viral genes and 66 transcription factors from enteric HAdVs, which aid to increase viral 67 mRNA levels and promote propagation of these fastidious human 68 adenoviruses in cell culture (Kim et al., 2010). This cell line has only 69 been tested with laboratory reference strains and clinical isolates, 70 and its utility in detecting HAdVs from environmental samples has 71 not yet been determined. 72 The goal of this study was to test the feasibility of using the 73 improved cell line (293 CMV) for detecting enteric HAdVs, using 74 environmental sewage and water samples collected from differ75 ent surface water and wastewater sources. Detection of enteric 76 adenovirus was compared between the standard (G293) and the 77 improved transactivated (293 CMV) cell lines. For this study, the 78 replication of HAdV in the cell line was determined by mea79 suring the production of viral mRNA and determining the levels 80 of viral DNA; therefore, the results are representative of what 81 could be expected when using this improved cell line for either 82 a cell culture–qPCR or a cell culture mRNA RT qPCR approach. To 83 our knowledge, this is the first report using this improved cell 84 line for detecting infectious adenovirus from environmental water 85 samples. 86 49
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2. Materials and methods 2.1. Adenovirus type 41 stock The adenovirus type 41 stock (ATCC, VR-930) was propagated using the G293 host cells in 150 cm2 tissue culture flasks and purified by PEG as described by Rodríguez et al. (2013). The viral titer was determined using the mRNA qRT-PCR method described by Rodríguez et al. (2013).
2.2. Sewage samples Sewage samples were collected from three wastewater treatment plants (WWTP) in North Carolina. One liter was collected and concentrated, using organic flocculation, and polyethylene glycol precipitation (PEG) to a final volume of 5 mL as described by Rodríguez et al. (2013). To kill any bacteria and remove soluble impurities, 5 mL of chloroform was added to the sample and mixed to emulsify by inverting the tubes vertically for one minute. The samples were centrifuged (3000 × g, 20 min, 4 ◦ C). The supernatant was recovered, without disturbing the thin film layer above the chloroform, yielding a final sample volume of 5 mL. The supernatant was stored at −80 ◦ C and later assayed for viruses.
Samples were collected from six geographically distinct water treatment plants, within the United States, during three different sample times (September 2009, November 2009, and February 2010) per plant, yielding 18 samples. Each facility collected 24 L of raw source water. Samples were stored overnight at 4 ◦ C before shipping, by next day air express, to our laboratory. The temperature of the samples was recorded using a data logger system and never surpassed 7 ◦ C. Upon arrival at the UNC laboratory, 20 L samples were concentrated using hollow fiber ultrafiltration (HFUF), polyethylene glycol precipitation (PEG), and ultracentrifugation procedures in succession to a final volume of approximately 5 mL. The concentrated samples were stored in −80 ◦ C for future cell culture analysis. Twenty-liter volumes of source water were initially concentrated using tangential flow HFUF with a Hemoflow F80A sterile ultra-filter cartridge (molecular weight cut-off 15,000 to 20,000). The source water was re-circulated using a peristaltic pump at pressure between 15–25 psi until the recirculating water volume was emptied in the cubic polyethylene container holding the initial sample. To recover any viruses attached to the filter, the viruses were eluted by adding 250 mL of HFUF eluting solution (1XPBS/1% Laureth-12) consisting of 10 g/L of Laureth-12 and 50 L antifoam-A per 1 L of PBS (pH 7.4). The resulting volume of retentate was approximately 200–300 mL. The retentate was concentrated further using PEG precipitation at a final concentration of 9% of PEG 8000 (Fisher, Cat no. 233-1, or equivalent) and 0.3 M NaCl; the sample was separated into 150 mL volumes using 250 mL conical centrifuge bottles. The bottles were placed on an orbital shaker and allowed to shake overnight (200 rpm, 4 ◦ C). The next day, the bottles were centrifuged (5400 × g, 1 h, 4 ◦ C). The supernatant was decanted and pellets were resuspended using 15 mL of PBS (pH 7.5). The resulting water sample concentrates were combined and stored in a 50 mL polypropylene centrifuge tube. To kill any bacteria and to remove certain soluble impurities, 5 mL of chloroform was added to the concentrated sample from step 2 (PEG precipitation) and mixed to emulsify by inverting the tubes in vertical movement for one minute. The samples were centrifuged (3000 × g, 20 min, 4 ◦ C), the supernatant was recovered from the top without disturbing the thin film layer above the chloroform, and the volumes were recorded. The supernatant was stored at −80 ◦ C or ultracentrifugation immediately followed. The volume of the supernatant recovered after chloroform extraction (in the previous step) was adjusted to 50 mL by adding PBS (pH 7.5). The sample was transferred to a 75 mL ultracentrifuge tube and centrifuged (100,000 × g, 4 h, 4 ◦ C). Ultracentrifugation separated the viruses from the buffer and further reduced the sample volume to 5 mL. The supernatant was immediately decanted and the pellet was resuspended in 5 mL of PBS (pH 7.5). The concentrated samples were aliquoted in four 1.2 mL tubes and stored at −80 ◦ C for subsequent virus infectivity assay. 2.4. Cell culture infections and detection of adenovirus mRNA and DNA during cell culture infectivity assays The infectivity assays were performed as described by Rodríguez et al. (2013). A 1.5 mL inoculum was produced by diluting 350 L of concentrated sample with 1050 L complete MEM medium, without serum and containing 10 g kanamicin, 50 g gentamicin, and 20 g nystatin per mL of medium. The use of media in the inocula reduced cell detachment from their substrate after inoculation. After one hour of incubation at 37 ◦ C for viral adsorption, the inoculum was removed, 6 mL of complete MEM medium with 2% fetal bovine serum was added to each flask, and the cell cultures were incubated at 37 ◦ C in a water-jacked CO2 incubator.
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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2.5. Detection of adenovirus mRNA and DNA during cell culture infectivity assays
Detection of adenovirus mRNA in cell culture infectivity assays 173 was performed as described by Rodríguez et al. (2013). The nucleic 174 acids from the cell monolayers inoculated with concentrated water 175 samples were extracted and purified, as described by the manu176 facturer of the Qiagen RNeasy kit (Qiagen, Valencia, CA). The final 177 nucleic acid extract was split into two 25 L volumes. One volume 178 was treated with DNAse (Promega, Madison, WI) to remove the 179 DNA from the sample and used for mRNA analysis. The second 180 untreated aliquot was used for quantifying the adenovirus DNA. 181 The reverse transcription step was performed using the M-MLV 182 reverse transcriptase (Invitrogen, Carlsbad, CA) and the oligo dT18 183 primer, as described previously (Rodríguez et al., 2013). The cel184 lular housekeeping gene, GAPDH, was used as an internal control 185 for the mRNA assay. Real-time PCR for the quantification of cDNA 186 and viral DNA used the primer-probe set specific for the hexon 187 gene of human adenovirus (species A to F) as published by Jothiku188 Q4 mar et al. (2005). The real-time PCR assay was performed using 189 the QuantiTeck probe PCR master mix (Qiagen, Valencia, CA). The 190 concentration of the primers and dual-labeled probe were 1.0 M 191 and 0.1 M, respectively. The real-time PCR was run in a SmartCy192 cler (Cepheid, Sunnydale, CA) and the cycling conditions were as 193 follows: initial denaturation for 15 min at 95 ◦ C and then 45 cycles 194 of the following: 15 s at 95 ◦ C, 30 s at 58 ◦ C, and 15 s at 72 ◦ C. The 195 control used for calibration was created by inserting a fragment of 196 the adenovirus hexon gene into a TOPO clone as described previ197 ously (Rodríguez et al., 2012). The PCR efficiency was 110% and the 198 calculated detection limit was 1.5 genome copies per PCR reaction 199 (2 L assayed). Ct values were transformed into log10 copies/PCR 200 reaction and then the concentration was adjusted per 50 L of final 201 volume of nucleic acid obtained after the purification of the viral 202 contents of a 25 cm2 flask. 172
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2.6. Comparison of transactivated 293 CMV and standard G293 cell lines for the detection of HAdV 41 and HAdV from sewage and source water samples To demonstrate the performance of the transactivated cell line to promote the growth of enteric HAdVs, two different HAdV 41 concentrations were used, 50 and 5 infectious units, respectively. All the assays were performed in parallel and the propagation of HAdVs was confirmed using real-time RT-PCR targeting the adenovirus hexon gene. The concentrations of HAdVs in sewage samples were determined using the combined cell culture and real time RT-PCR assay described previously for detecting adenoviral mRNA (Rodríguez et al., 2013). Comparison between cell lines for adenovirus detection by infectivity assays were performed, in parallel, using a purified concentrated sewage sample as inoculum and adjusting to adenovirus concentrations of 10 infectious units (IU), 2 IU, and 0.8 IU per well. Adenovirus propagation was determined by measuring viral mRNA and viral DNA at 2 days post infection (DPI), 3DPI, and 5DPI as described later in the manuscript. To compare the growth of different concentrations of HAdV-41 stock and sewage samples, 3 cell culture flasks per sample were used. Detection of HAdV from surface water by each cell line was conducted for 18 surface water samples as described before. Concentrated sample aliquots of 350 L were mixed with 1 mL of MEM with no BSA, and this mixture was used as inoculum for cell culture infection. Five 25 cm2 flasks containing the cell monolayer were used per sample. In the case of standard G293 cell line, six flasks per sample were used with one flask used to test cell toxicity of the sample inoculum. For the 293 CMV cell line, the flasks were incubated for three days and for the G293 cell line, the flask were incubated for five days. The different incubation times corresponded to optimal
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times required for viral nucleic acid detection as established previously. Adenoviruses from positive flasks were typed by sequencing and amplifying a portion of the hexon gene using the primers hex1 and hex2 described by Ko et al. (2003). 2.7. Statistical analysis An analysis of variance (ANOVA) was used to compare the expression of viral mRNA and concentration of viral DNA during cell culture propagation in the two cell lines (G293 and 293 CMV). For this analysis, the concentrations were transformed into log10 units. Chi square analysis was used to compare the frequency of detection of infectious HAdVs from source water samples between the two cell lines, G293 and 293 CMV. For this comparison, the detection of viral mRNA or viral DNA was used as indication of infection, and the results were expressed as flasks either positive or negative for viral replication. Each concentrated source water sample was analyzed with each cell line in parallel, as previously described. All statistical analyses were performed using the Minitab program (release 14.23, Minitab Inc., State College, PA). 2.8. Quality assurance and control Samples of source water were cooled at 4 ◦ C and shipped overnight in coolers along with a chain of custody form, which was kept for each sample. The sample sheet, custody form, supporting information, and results were archived. The water samples (from WTPs) and sewage samples (from WWTPs) received in NC were processed and analyzed in a lab room dedicated specifically for field samples. The room was used exclusively for processing field samples and no work with positive controls, microbial reference strains, or their nucleic acids was performed in that room. Nucleic acid extractions and assays of samples in cell culture were also performed within the field samples room. Positive controls were processed in a separate lab room, which was dedicated for working with known pathogens and involved the use of positive control and reference strains. The different stages in the analysis of water samples was done on a schedule such that all samples being processed were at the same stage of analysis and no work at different stages of analysis was performed simultaneously. The analysis of PCR product was the last step and was conducted in a separate lab room designated for PCR amplifications and related PCR product analysis. All equipment and reusable labware was autoclaved or decontaminated with sodium hypochlorite, depending on the nature of the equipment and labware. 3. Results 3.1. Comparison of the performance of the new transactivated 293 CMV cell line with the standard G293 cell line for the propagation of adenovirus 41 The propagation of HAdV 41 was compared between the G293 and 293 CMV cell lines. The expression of the viral hexon gene was consistently higher in the 293 CMV cell line than in the G293 cell line, but the magnitude of difference declined with longer incubation time (Fig. 1). The difference observed in mRNA levels per cell line was statistically significant (p = 0.01). 3.2. Comparison of the transactivated 293 CMV cell line with the standard G293 cell line for the detection of human adenovirus in concentrated sewage samples The propagation of HAdV from the collected sewage in the new transactivated 293 CMV cell line at very low concentrations was
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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the incubation time is longer, the time to maximum detection by the 293 CMV cell line is earlier. The results for the WWTP-3 samples yielded mostly non-detectable values; therefore, this sample was not used for quantitative comparison. Positives flasks were analyzed by sequencing and most of the adenoviruses detected were from subgroup F. Adenoviruses subgroup A were detected but in only 2 flasks. No apparent differences were observed in which adenovirus types were detected between the improved and standard cell lines.
3.3. Comparison of the G293 cell line with the 293 CMV cell line for the detection of infectious human adenovirus in source water sample concentrates
Fig. 1. Comparison of transactivated 293 CMV (䊉) cells with G293 cells () for the propagation of adenovirus 41 as measured by the production of viral hexon gene mRNA at initial viral inocula of 5 infectious unit (100 genome copies) per flask and 50 infectious units (1000 genome copies) per flask. The titer of the stock was determined both by direct PCR and by infectivity assay. Reported concentrations are the average of three experiments (n = 3) and the units are log10 copies per well; error bars represent standard deviation.
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demonstrated, as shown in Table 1. Significantly higher concentrations of viral mRNA (p = 0.015) and viral DNA (p = 0.045) were observed in the 293 CMV monolayers than in the G293 monolayers. WWTP-1 samples demonstrated greater numbers of positive samples detected compared to other sample sources, when targeting viral hexon gene mRNA and viral hexon gene DNA for both cell lines. The infectivity assays conducted for HAdVs in WWTP-2 yielded detectable levels of both viral mRNA and viral DNA in the new transactivated 293 CMV cell line as early as 2 DPI. For the standard G293 cells, HAdVs were detected only at 5 DPI. Notably, the viral DNA level detected for G293, which was the only day with detectable results, falls within the range of detectable values for 293 CMV cells. Although the G293 cell line eventually reaches similar levels of adenovirus hexon gene mRNA detection as in the 293 CMV cell line if
Source water samples from different locations in the US were concentrated by the successive steps of HFUF, PEG precipitation, and ultracentrifugation and used to compare human adenovirus detection in the transactivated 293 CMV and G293 cell lines. Initial testing was conducted with concentrated WTP source water samples to determine possible interferences from sample cytotoxicity. Concentrated sample 2 from WTP-E showed high toxicity that manifested as cell death; therefore, this sample was further diluted two-fold with maintenance medium (no serum) prior to performing the infectivity assays as this dilution reduced the toxicity of the sample. All other concentrated samples did not show toxicity when inoculated at 350 L concentrated sample volumes per cell culture. All WTP source waters were subsequently analyzed using the 293 CMV cell line (Table 2). The inoculated cells were incubated for 3 days as previous results showed the highest expression of viral hexon gene nucleic acid was observed at this incubation time for 293 CMV cells. The equivalent volume of source water analyzed for each sample was 5 L, except for the second sample collected from WTP-E, for which the sample volume was reduced to 1.6 L of source water per sample in order to reduce cell toxicity. Overall, 8 source water samples were positive for infectious adenoviruses hexon gene nucleic acid using the transactivated 293 CMV cell line, and 4 samples were positive using the G293 cell line, of all 18 source water samples collected and analyzed, as shown in Table 2. Three samples were positive with both cell lines, while
Table 1 Comparison of 293 CMV and G293 cell lines for the propagation of human adenoviruses in sewage samples from three different waste water treatment plants (WWTPs). Sewage sample
1
Initial adenovirus concentration Infectious units (IU)a
Genome copies (GC)b
11
240
G293 cell line
CMV
STD
2
2
44
CMV
STD
3
0.8
11
CMV STD
DPIc
2 3 5 2 3 5 2 3 5 2 3 5 3 5 3 5
Adenovirus replication outcome Viral mRNA (total 3 wells)
Viral DNA (total 3 wells)
Positive
Log10 GC (±STD)
Positive
Log10 GC (±STD)
3 3 3 3 3 3 2 3 2 0 0 2 0 1 0 0
3.1±0.1 3.5±0.3 2.6±0.2 3.0±0.0 2.4±0.6 1.9±0.9 1.7 2.5±0.8 1.6 NDd ND 1.6 ND 0.7 ND ND
3 3 3 3 3 3 2 3 3 0 0 3 0 2 1 2
3.4±0.1 4.1±0.3 4.7±0.4 2.5±0.8 3.5±0.8 4.5±0.0 1.9 3.2±0.7 3.6±0.7 ND ND 3.5±0.6 ND 2.2 1.3 1.6
a Infectious units per flask used in the inoculum were determined using mRNA reverse transcription PCR in infected G293 cell lines as described previously (Rodríguez et al., 2013). b Genome copies per flask used in the inoculum were determined using real-time PCR as described previously (Rodríguez et al., 2013). c Days post infection. d None detected after 45 cycles.
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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Table 2 Comparison of results for human adenovirus infectivity assays of source water samples between the standard (G293) and improved (293 CMV) cell lines. Sitea
A
B
C
D
E
F
293 CMV
Sampleb
1 12 18 2 11 17 3 7 15 4 9 16 6 8 13 5 10 14
Total
G293
mRNAc (positive/total)
DNAd (positive/total)
Combined resultse
mRNA (positive/total)
DNA (positive/total)
Combined results
2/5 0/5 0/5 0/5 1/5 0/5 0/5 2/5 2/5 1/5 2/5 0/5 0/5 1/5 1/5 0/5 0/5 0/5 13/90
1/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 1/5 2/5 0/5 0/5 1/5 1/5 0/5 0/5 0/5 8/90
Positive Negative Negative Negative Positive Negative Negative Positive Positive Positive Positive Negative Negative Positive Positive Negative Negative Negative 8/18
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 1/6 0/6 2/6 0/6 0/6 0/6 1/6 0/6 0/6 0/6 4/108
0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 1/6 0/6 0/6 1/6 0/6 0/6 2/6 0/6 0/6 0/6 4/108
Negative Negative Negative Negative Negative Negative Negative Negative Positive Negative Positive Positive Negative Negative Positive Negative Negative Negative 4/18
a
Source water sample collection site (water treatment plant). Twenty liter source water sample. The sample numbers are in order of collection. Samples 1 to 6 were collected in September 2009, samples 7 to 12 were collected in December 2009, and samples 13–18 were collected in March 2010. c Detection of adenovirus mRNA in cell monolayers after cell culture infection. The analysis times for 293 CMV cell lines were performed after 3 days post-incubation and for G293 cell line after 5 days post-incubation. d Detection of adenovirus DNA in cell monolayers after cell culture inoculation and incubation. Time of analysis for viral DNA was the same as for mRNA. e Cell culture result per sample, based positivity for DNA or mRNA scored as positive. b
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1 sample was positive when analyzed with the G293 cell line and negative with the 293 CMV cell line. Five samples were positive when assayed with the 293 CMV cell line and negative when analyzed with the G293 cell line. More samples were positive within the 293 CMV cell line as compared to the G293 cell line; however, the increase in samples positive for virus detection was not significant as determined by chi square analysis (p > 0.1). For this analysis, a sample was considered positive when one of the flasks was positive by detecting either the viral mRNA or viral DNA. However, the number of cell culture flasks positive for HAdV as determined by presence of either viral mRNA or viral DNA, was significant with more flasks positive for the 293 CMV cell line (21 of 90) than the G293 cell line (8 of 108) (Table 2, p = 0.002).
4. Discussion The evaluation of the new transactivated 293 CMV cell line for the detection of wild type adenoviruses viruses at concentrations encountered in environmental samples is important evidence for demonstrating the utility of the improved cell line in monitoring the presence of adenovirus in the water environment. This study demonstrated the effectiveness of this new cell line for the detection of infectious HAdV from sewage and source water samples. The use of an improved cell line resulted in an earlier increase in viral replication when assaying concentrated sewage samples and more positive source water samples at shorter incubation times. The transactivated 293 CMV cell line was created from the parent G293 cell line, which is a cell line containing a portion of the HAdV 5 genome that is used commonly for the propagation of enteric adenovirus. This initial genetic modification resulted in a cell line capable of supporting efficient growth of enteric HAdV (Graham et al., 1977). However, enteric HAdV shows a variable and inconsistent manifestation of viral cytopathogenic effects in the G293 cell line. The inconsistent onset of CPE of enteric HAdV in cell culture is mostly due to the poor expression of early genes, the inability to evade the cell immune response, and the delay in the release of the mature viral particles (Brown et al., 1992; Mautner et al.,
1999). For example, interferon deficient cells created by expressing the V protein of paramyxovirus simian virus 5 resulted in better propagation of enteric HAdV (Sherwood et al., 2007). The 293 CMV cell line contains the gene to produce transactivated protein IE1 from cytoglomegavirus that promotes the expression of HAdV early genes, resulting in higher levels of viral mRNA and viral DNA when compared to the G293 cell line (Kim et al., 2010). As demonstrated in the present study, there was increased earlier expression of HAdV mRNA at higher levels in the 293 CMV cell line than in the G293 cell line, resulting in faster detection of HAdV isolates from environmental samples. The assessment of the transactivated cell line in this work was designed to demonstrate the early detection of human adenovirus at very low concentrations, particularly of environmental strains (wild types), determine the time required for detection of environmental adenovirus and document the ability of the transactivated cell line to monitor the presence of infectious HAdVs in environmental samples. Sewage samples were used to determine the detection and propagation of adenovirus at concentrations that are relevant to analysis of environmental samples. A sewage sample (number 2) showing no HAdV detection in the first two incubation times for the standard cell line resulted in HAdV detection as early as 2 days for the improved cell line (Table 1). Because infectivity assays in both cell lines were performed in parallel using the same inoculum and internal control, these results provide evidence that the improved cell line is capable of earlier detection of human adenovirus. However results observed in another sewage sample (number 3) were less conclusive because the concentrations of adenovirus in those assays were almost below the detection limit of one infectious unit per flask and therefore would require more replicate assays to detect differences in HAdV detection by the two cell lines (Rodríguez et al., 2013). Improving infectivity assays for enteric adenoviruses is challenging because environmental isolates tend to have different growth kinetics than corresponding laboratory-adapted isolates. For example, during the development of a plaque assay for enteric adenovirus, multiple isolates were tested and selected for their capacity to produce CPE (Cromeans et al., 2008). After selection and Q5
Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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several passages in cell cultures isolates producing stable plaques were obtained, making this assay specific for the selected enteric adenovirus strain. This assay for the cell-culture adapted HAdV has been useful for studying the stability of the virus in the environment (Rigotto et al., 2011) and could be useful for determining disinfection performance and the performance of recovery methods. However, because this plaque assay is isolate-specific, its applicability to the detection of wild type infectious adenovirus found in the environment may be limited. Other improved cell culture assays for adenovirus are based on detecting viral proteins during cell culture propagation using the 293 cell line and flow cytometry. This approach has been able to quantify human adenovirus from environmental samples (Li et al., 2010). Initial assessment of the transactivated cell line 293 CMV was performed using laboratory adapted strains of enteric adenovirus and clinical strains obtained from stool samples (Kim et al., 2010). The use of a clinical strain of adenovirus 40 helped demonstrate the applicability of the transactivated cell lines to non-cell culture adapted viral isolates, but this initial assessment was performed using higher viral titers not relevant to the much lower viral titers of most environmental samples (Kim et al., 2010). A new and improved 293 cell line (293 RAS) that overexpresses the RAS protein has also been developed that has demonstrated enhanced capability to propagate enteric adenovirus at low concentrations (Si et al., 2013). Comparison experiments between the 293 CMV and the 293 RAS cell lines to detect wildtype human adenoviruses from environmental samples are now warranted. HAdV has been proposed for monitoring human fecal contamination in water because of its continuous presence in sewage samples and its detection in fecally contaminated waters (Silva et al., 2011). Another advantage of the detection of HAdV in sewage and sewage-contaminated waters is its strict human origin, as it has not been detected in any other host besides humans (Ahmed et al., 2010)., Despite previous attempts to use different cell culture approaches (Jiang, 2006), determining HAdV infectivity in environmental samples is challenging. The detection of HAdV infectivity in environmental samples using the onset and appearance of cytopathic effects in the cell monolayer has been reported previously (Jiang et al., 2009). However, the detection of viral replication intermediates, such as the approach demonstrated in this study for early detection of viral mRNA, can result in faster detection because it is not necessary to wait for the onset of viral induced cytopathic effect (Ko et al., 2003). In addition to a previous study on detecting HAdV in environmental samples by combined cell culture, qPCR and mRNA RT-qPCR in 293 cells (Rodríguez et al., 2013), Ogorzaly et al. (2013) have also reported the detection of HAdV DNA by qPCR in environmental samples using a sub-clone of 293 cells, called 293A. It should be noted, their study design focused on only DNA to detect HAdV and not RNA, as done in the protocol. Their results for detecting HAdV DNA are similar to those described above, in that detection of HAdV DNA was possible within 2 days and did not increase further by 3 days. However, it is not possible know if the cell culture infectivity protocol and 293 cell line used in their study and in the current study are comparable or differ in performance unless compared in parallel for the same viral targets of mRNA and DNA on the same environmental samples simultaneously, as done in comparing standard 293 cells to 293 CMV cells. Such further comparisons are recommended. Similarly, the appearance of viral DNA (Chapron et al., 2000; Dong et al., 2010) or viral early proteins (Calgua et al., 2011; Li et al., 2010) also have been targets for more rapid detection of HAdVs than the visual appearance of CPE in other studies. The improved transactivated 293 CMV cell line could be adapted to any of these approaches for infectivity detection of early viral replication products, possibly improving their performance and decreasing detection time.
In the present study, detecting infectious HAdV by detecting the viral mRNA using the 293 CMV cell line resulted in better detection (greater numbers of positive cell cultures) and shorter incubation times than possible with the standard G293 cell line. In addition, as demonstrated by Kim et al. (2010), onset of cytopathic effect on the 293 CMV cell line is clearer than in the standard G293 cell line. Therefore, the 293 CMV cell line may better facilitate the development of plaque assays that detect infectious enteric HAdV in environmental samples. In the analysis of source water samples, greater numbers of flasks were positive by analyzing the production of adenovirus mRNA during cell culture than analyzing for the viral DNA. This may be because more copies of mRNA per DNA are produced during early stages of viral replication in cell culture infectivity assays (Ko et al., 2003). However, as infection progresses, mRNA levels remain constant and DNA levels increase, as was observed in the results of Table 1. Therefore, longer incubation time could have resulted in the detection of viral DNA in samples where only mRNA was detected initially. Although the nucleic extraction method used in this experiment was optimized for extraction of mRNA, detection of viral DNA in the experiments with sewage samples was consistent with the level of inoculum and incubation time, demonstrating that this method also was efficient for the recovery of viral DNA. In conclusion, the results of this study indicate that the new transactivated 293 CMV cell line demonstrated improved ability to rapidly detect low levels of infectious human adenoviruses from sewage and environmental water samples than the standard G293 cell line. In addition, the use of transactivated 293 CMV cells resulted in faster replication of human adenoviruses found in environmental sewage and water samples when compared to the G293 cells. This conclusion is based on earlier detection of greater amounts of viral hexon gene mRNA by a sensitive and specific quantitative RT-PCR assay applied to nucleic acid extracts of infected cell cultures. The results of this study also provide evidence that the new 293 CMV cell line may detect more infectious human adenoviruses than did the standard G293 cell line in environmental source water samples. Use of this new 293 CMV cell line has the potential to provide more rapid and potentially increased detection of infectious human viruses in environmental water and wastewater samples; thereby, improving monitoring and surveillance of infectious human enteric adenoviruses in these environmental waters for more timely management decisions and response actions. Acknowledgments This work was funded by the Water Research Foundation Q6 (Project Number 3181) and the Agriculture Research Center pro- Q7 gram of the Ministry for Food, Agriculture, Forestry and Fisheries, Korea. We would like to thank to the facility managers of the water and wastewater treatment plants for agreeing to participate in this project and providing environmental samples. References Ahmed, W., Goonetilleke, A., Gardner, T., 2010. Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia. Water Res. 44, 4662–4673. Aslan, A., Xagoraraki, I., Simmons, F.J., Rose, J.B., Dorevitch, S., 2011. Occurrence of adenovirus and other enteric viruses in limited-contact freshwater recreational areas and bathing waters. J. Appl. Microbiol. 111, 1250–1261. Bofill-Mas, S., Calgua, B., Clemente-Casares, P., La Rosa, G., Iaconelli, M., Muscillo, M., Rutjes, S., Husman, A.M., Grunert, A., Graeber, I., Verani, M., Carducci, A., Calvo, M., Wyn-Jones, P., Girones, R., 2010. Quantification of human adenoviruses in European recreational waters. Food Environ. Virol. 2, 101–109. Brown, M., Wilson-Friesen, H.L., Doane, F., 1992. A block in release of progeny virus and a high particle-to-infectious unit ratio contribute to poor growth of enteric adenovirus types 40 and 41 in cell culture. J. Virol. 66, 3198–3205.
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Please cite this article in press as: Polston, P.M., et al., Field evaluation of an improved cell line for the detection of human adenoviruses in environmental samples. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.05.002
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