key: cord-0301462-mul0cukp
authors: McLellan, N.L.; Weir, S.C.; Lee, H.; Habash, M.B.
title: Polyethylene glycol (PEG) methods are superior to acidification for secondary concentration of Adenovirus and MS2 in water
date: 2021-11-20
journal: bioRxiv
DOI: 10.1101/2021.11.19.469352
sha: da4933716cdf27af2a7a1b18d399fc76d4b81bc7
doc_id: 301462
cord_uid: mul0cukp

Enteric viruses are a leading cause of waterborne illness worldwide and surveillance studies lack standardization in method selection. The most common and cost-effective approach to concentrating viruses from water samples involves virus adsorption and elution (VIRADEL) procedures, followed by secondary concentration. There is a lack of consistency in how secondary concentration methods are practiced and some methods may have better recovery for particular groups of viruses. Secondary concentration methods typically involve precipitation and the most common methods employ organic flocculation (OF) by acidification at a pH of 3.5, or precipitation by polyethylene glycol (PEG) in combination with the addition of NaCl. In this study, the recovery of coliphage MS2 using the plaque assay and human adenovirus strain 41 (HAdV41) using cell-culture and qPCR assays were evaluated by OF and PEG secondary concentration of spiked samples of wastewater, surface water, and groundwater. The recovery of MS2 and HAdV41 by PEG precipitation was significantly higher than that by OF (p<0.0001) when viruses were detected by culture based methods and marginally better when HAdV41 was enumerated by qPCR (p<0.019). The recovery of HAdV41 by qPCR ranged from 75.3% to 94.4% (n=36). The mean recovery of MS2 by OF was 4.4% (0.9%-7.7%; n=14) and ranged from 57.1% to 87.9% (n=28) for the PEG methods. The poor recovery of MS2 by OF was attributed to inactivation or poor stability at acidic conditions as MS2 were not recovered in the supernatant following OF and centrifugation. The inconsistency and lack of justification for method selection in many studies calls for a systematic study to inform guidance and standardization with respect to the application of concentration methods for various water types and viral pathogens. IMPORTANCE MS2 should not be used as a process control for methods involving acidification and culture-based detection. The dense floc produced by the PEG method may have contributed to higher recoveries as the pellet was more compact and stable than the loose pellet formed by OF. Standard methods for the detection of enteric viruses and surrogates that involve acidification could be modified with PEG precipitation to uphold virus recovery and minimize inactivation.

groundwater. The recovery of MS2 and HAdV41 by PEG precipitation was significantly higher 23 than that by OF (p<0.0001) when viruses were detected by culture based methods and marginally better when HAdV41 was enumerated by qPCR (p<0.019). The recovery of HAdV41 25 by qPCR ranged from 75.3% to 94.4% (n=36) . The mean recovery of MS2 by OF was 4.4% (0.9%-26 7.7%; n=14) and ranged from 57.1% to 87.9% (n=28) for the PEG methods. The poor recovery of 27 MS2 by OF was attributed to inactivation or poor stability at acidic conditions as MS2 were not 28 recovered in the supernatant following OF and centrifugation. The inconsistency and lack of 29 justification for method selection in many studies calls for a systematic study to inform 30 guidance and standardization with respect to the application of concentration methods for 31 various water types and viral pathogens. 32 IMPORTANCE 33 MS2 should not be used as a process control for methods involving acidification and 34

culture-based detection. The dense floc produced by the PEG method may have contributed to 35 higher recoveries as the pellet was more compact and stable than the loose pellet formed by The detection of viruses in environmental waters (e.g. groundwater, surface water, and 44 wastewater) typically requires isolation and concentration methods due to low titers and 45 methodological sensitivity of detection methods. Nevertheless, low concentrations of viruses 46

can pose a human health risk as the infectious dose of many viruses is low (e.g. 1 to 10 virions) 47

(1, 2). Standard methods for the detection of enteric viruses in the environment are limited and 48 many are not adaptable for all human viruses of concern or viral surrogates (e.g. phage); yet 49 they are required to inform public health protection measures (3). 50

Procedures for detecting waterborne viruses typically begin with isolating viruses from 51 the bulk water (e.g. groundwater or surface water) either by size-exclusion with ultrafiltration 52 or with VIRADEL (virus adsorption-elution) methods (4-6). VIRADEL methods are most 53 commonly employed due to costs and access to instrumentation. The limitations associated 54 with these isolation and "primary concentration" methods for viruses from water have been 55 reviewed elsewhere (5). The eluted suspension from a charged ultrafiltration and VIRADEL 56 methods often requires secondary concentration prior to employing a culture-or molecular-57 based method for viral detection and quantification. Secondary concentration may be 58 performed by a variety of methods, though the most common methods include organic 59 flocculation (OF) by acidification and precipitation by polyethylene glycol (PEG) (5-8). Some 60 matrices have elevated concentrations of viruses such as wastewaters, and these do not 61 typically require the initial isolation step. Higher-titer samples can be concentrated directly by a 62 "secondary concentration" method.

There is a lack of consistency in the application of secondary concentration methods 64 with respect to: (a) the type of method selected and (b) the execution of each type of method. 65

For example, PEG is often applied in combination with various molar concentrations of NaCl 66 ranging from 0.2 to 1.5 M, and the incubation period may range from less than 1 h up to 24 h 67 (7-13). The USEPA standard Method 1615 for the detection of enterovirus and norovirus in 68 water employs OF by acidification for secondary concentration and recommends the use of 69 poliovirus (Sabin poliovirus 3) as the process control virus (PCV) (6). The standard procedure for 70 OF requires acidification of the suspension from an initial pH of about 9.0 (of the buffered beef 71 extract used for elution of a cartridge filter) down to pH 3.5 ±0.1. Poliovirus has been shown to 72 be resistant to acidification and drastic changes in pH. Huang et al. (2000) observed that 73 poliovirus 1 was not inactivated by pH changes between 3.5 and 9.5 (14). The resistance of 74 poliovirus raises the question as to its suitability as a PCV for virus detection methods; 75

particularly if the methods are adapted for the detection of other, less stable, viruses. 76

Human adenoviruses (HAdV) have been proposed as a suitable surrogate for 77 determining viral contamination in source waters due to their stability in environmental waters 78 and persistence (5, (15) (16) (17) (18) (19) (20) . However, standard methods for the detection of HAdV are lacking. 79

Further, the F-specific coliphage MS2 is commonly employed as a viral surrogate for 80 understanding the fate and transport of viruses in the environment and for performance 81 demonstrations of water treatment processes (e.g. ultraviolet light disinfection) (21) (22) (23) (24) (25) . 82

However, studies have indicated lower stability of some viruses during pH changes and in acidic 83 environments (e.g. pH < 4), including . 84

The present study describes the recovery of MS2 and a human strain of adenovirus 85 (HAdV41) from surface water, groundwater and wastewater by two secondary concentration 86 methods: OF and PEG precipitation. Additionally, two molar concentrations of NaCl were trialed 87 with PEG precipitation as varying concentrations have been cited in previous studies and to 88 evaluate if this parameter is associated with improved recovery and resulting detection of 89 viruses of interest (5, 8, 12, 14) . 90

Raw groundwater was collected from a municipal well deemed as groundwater under 93 the direct influence (GUDI) of surface water and had a turbidity of 0.16 NTU, temperature of 94 2.1 o C, conductivity of 712 µS/cm, and absent of E. coli and total coliform detections at the time 95 of collection. The raw surface water sample was collected in winter from the Grand River 96 watershed which is heavily impacted (agriculture and urban land uses) and had a turbidity of 97 10.1 NTU, temperature of 4.2 o C, conductivity of 577.13 µS/cm, E. coli concentration of 1.9x10 3 98 CFU/100 ml, total coliform concentration of 9.1x10 4 CFU/100 ml at the time of collection (30). 99

Raw wastewater was collected from a municipal supply. Conductivity of the prepared 1L water 100 samples of wastewater, concentrated surface water, and concentrated groundwater were 117, 101 61, and 124 µS/cm, respectively. Background concentrations of MS2 in the prepared 1L samples 102 of wastewater, concentrated surface water, and concentrated groundwater were 5.6 log PFU/L 103 (±5.3 log PFU/L), 4.1 log PFU/L (±4.1 log PFU/L), and 1.3 log PFU/L (±1.0 log PFU/L) where n=3 104 for each enumeration. Therefore, the spiked MS2 concentration target was >6 log MS2 PFU per

The mean % recovery of MS2 PFU by OF was 4.3% (range of 1.0% to 7.6%; n=12) and 129 was between 61 and 90% by the PEG methods. The recovery of MS2 by OF was significantly 130 lower than that of the PEG method with either salt concentration (p<0.0001). There was no 131 significant difference found between the recovery of MS2 by the two PEG methods (p=0.976). 132

There was no significant difference between the recovery of MS2 from the various water types 133 (p≥0.210). While it may appear that the recovery by the PEG methods is more variable than 134 that of the OF method, the low recovery of OF may bias this observation. 135

There was a high degree of variation in the recovery of HAdV41 infectious units (IU) 137 between replicates, concentration methods, and water type (FIG B) . The recovery of HAdV41 IU 138 by PEG methods performed on groundwater samples (range 19.5-51.3%; n=6) was lower than 139 that achieved for surface water (range 55.7-100.4%; n=6) and wastewater (range 71.8-102.2%; 140 n=6) samples. The recovery of HAdV41 IU by OF for all water types ranged from 6.9-50.7% 141 (n=9). For groundwater samples, there was no significant difference between the recovery of 142 OF and PEG methods for HAdV41 IU (p>0.674). There was no significant difference between the 143 recoveries of the PEG methods with different NaCl concentrations for any water type (p=0.482). 144

The recovery of HAdV41 IU was significantly higher by PEG with either salt concentration than 145 the OF method for surface water and wastewater samples (p<0.0001). 146

The recovery of HAdV41 enumerated by qPCR ranged from 75.3-94.4% by all secondary 148 concentration methods for all water types (FIG C) . The recovery for wastewater samples was significantly higher than that achieved for groundwater samples by both PEG methods 150 (p<0.003); though the variability was substantially lower than when HAdV41 was detected by 151 cell culture. 152 (iv) MS2 Recovery in Supernatants. 153 MS2 is a common PCV and was enumerated in the supernatant of each test to evaluate 154 the fate of MS2 that was not recovered in the pellet following secondary concentration by OF 155 and PEG methods. The spiked concentration of MS2 was used as a baseline to calculate the 156 average recovery of MS2 in the pellet, supernatant, and the resulting "unaccounted" fraction 157

which may indicate the inactivated MS2 not detected by the plaque assay; the results are 158 shown in Fig. 3 . The recovered fraction of MS2 in the supernatant ranged from 1.1% to 20% for 159 the PEG methods, and from 13.9% to 34.8% for the OF method tested. The unaccounted 160 fraction of MS2 from the PEG methods ranged from 5.9% to 29.1% for the PEG methods, and 161 from 58.7% to 80.9% for the OF method tested. The recovery of MS2 in the supernatant was 162 lowest, and the fraction of unaccounted MS2 was highest, in surface water samples for all 163 secondary concentration methods tested. 164

In this study, the recovery of MS2 and HAdV4 by PEG precipitation, when detected by 166 culture-based tests, was found to be superior to that of OF by acidification. The >90% loss of 167 culturable MS2 by OF is suspected to be primarily due to the inactivation of MS2, which is 168 known to have poor stability in acidic environments. Acidification has been reported to impact 169 bacteriophage types using 2-10% solutions of PEG6000 (34). Thereafter, Lewis and Metcalf (1988) 185 found that PEG precipitation was more effective than OF for the concentration of rotaviruses 186 (WA and SAII) and hepatitis A virus (HAV) from estuarine and fresh waters (11). PEG6000 was 187 added to each sample (8%) and stirred for 1.5 h at 4 o C, followed by centrifugation at 10,000 g 188 for 20 min. The pellet was reconstituted in phosphate buffer solution (PBS). Various 189 concentrations of PEG6000 were tested (0, 8%, 12%, 15%, 20% w/v), and between 12-15% w/v 190 was found to be optimal for viral suspensions. Nevertheless, some studies continue to perform 191 PEG methods with PEG concentrations lower than 12% w/v (9, 28), and others have found that higher PEG concentrations can enhance the recovery of some viruses (e.g. TGEV and HAV at 193

The SARS-COV-2 pandemic has sparked a renewed interest in evaluating, optimizing, with that of non-enveloped MS2 by RT-qPCR and found that MS2 showed differences in 213 recovery and was not a suitable indicator to validate the extraction and recovery of enveloped viruses (42). This recent interest in evaluating various concentration methods has highlighted 215 the inconsistencies in how PEG methods are applied and the lack of standardization; as many of 216 these studies include or exclude pre-treatment steps, use a range of PEG concentrations (e.g., 217

up to 20% PEG (48)), and a range of incubation times from 4 h to "overnight". 218

This study used a higher concentration of PEG (12% w/v) than has been used in other 219 studies (5, 8). The recovery of viruses in this study are similar to, or greater than, those found 220 by others that used PEG concentrations of 10% or less (10, 28). Enriquez and Gerba (1995) 221 found no significant difference between the average recovery of HAdV40 (detected by cell 222 culture) in tap water, sea water, and wastewater (secondary sewage) by OF (38.6%) and PEG 223 precipitation (40%), where the PEG methods was performed with 7% PEG8000 and 0.5 M NaCl 224 incubated for 2 h at 4 o C (10). El-Senousy et al. (2013) found that PEG8000 (12%) with 1.5 M NaCl 225 provided significantly better (average 0.4 log better; p<0.05) recovery than OF for norovirus GI 226 and GII by RT-PCR from fresh produce and irrigation water (8). In another study, PEG was 227 applied at 14% w/v with 0.2 M NaCl to wastewater samples with overnight incubation at 4 o C, 228 the recovery of poliovirus detected by plaque assay was within an acceptable range of 59.5% 229 (±19.4%); though recovery of poliovirus was significant higher (106% ±1.6%) with an alternative 230 method employing skimmed milk acidification which requires a pH of 3 to 4 (7). Ye et al. (2016) 231 found the recovery of MS2 by PEG (8% w/v and 0.5 M NaCl) using a plaque assay was about 232 43.1% (±16.8%) during the concentration of municipal wastewater samples (28). 233

A combined approach, using acidification and PEG methods, has been used for 234 secondary concentration during surveillance of enteric viruses in environmental waters (49). 235 Farkas et al. (2018) acidified the beef extract elution from a VIRADEL primary concentration method to a pH of 5.5 and then held for 30 min followed by centrifugation to remove sample 237 particulates (2,500 xg, 10 min). The supernatant received 15% w/v PEG6000 treatment with 2% 238

NaCl with an overnight (16 h) incubation at 4 o C followed by centrifugation (10,000 xg for 239 30 min, 4 o C). This approach resulted in an average recovery of adenovirus gc by qPCR of about 240 60% from a river water source which was similar to the recovery of the process control virus 241 mengovirus of 59.5% (40.6 standard deviation). The authors suggest that the performance of 242 this method for a range of sample types (wastewaters, surface water, sediment, and shellfish 243 samples) could be standardized for routine monitoring. However, the necessity for the 244 acidification step is unclear and for many waters (lake and groundwater sources) the initial 245 centrifugation step would not be of benefit; in fact, it could contribute to additional losses of 246 particle-associated viruses. The recovery of HAdV41 gc was consistently >60% for all waters and 247 secondary concentration methods used in this study; therefore, the use of a hybrid method is 248 not recommended. 249 PEG methods have been performed with multiple washing steps of the pellet (50) which 250 may negatively affect the recovery of viruses. While efforts should be made to minimize the co-251 concentration of inhibitory compounds to qPCR and cell-culture assays with target viruses (51), 252 recovery should be upheld as a primary goal through viral concentration from environmental 253 water samples even when a process control is included. When PCR methods are employed, the 254 efficiency of nucleic acid extraction can be equally important to the concentration step, and a 255 review of commercial extraction kits has been reported by Iker et al. (52) . The dense floc produced by the PEG precipitation method in this study could contribute 272 to higher recoveries and higher consistency between replicates as the pellet was more compact 273 and stable than the pellet formed during OF. Further, PEG precipitation presents a more user-274 friendly method whereby the analyst can visually confirm that the suspension was effectively 275 centrifuged during the pelleting procedure and that the pellet is intact during decanting the 276 supernatant. A second centrifugation step (8000 xg for 5 min at 4 o C) to further compact the 277 pellet has been proposed to enhance virus recovery by flocculation methods, though the 278 benefits to recovery from this additional step have not been confirmed (9). In this study, the 279 supernatant was extracted by pipette, rather than by decanting or physical pouring. This approach allowed the centrifuge container to remain stable and caused minimal disturbance to 281 the pellet. 282

The results from this study suggest that the recovery of HAdV41 nucleic acid may be 283 improved for samples with higher turbidity or solids content as higher recoveries were achieved 284 for the wastewater sample, followed by the surface water and groundwater sample; while the 285 recovery of MS2 was not substantially impacted by water type. The particulate matter in more 286 turbid samples may provide a de facto flocculant-aid that provides greater surface area or 287 bridging to enhance the formation of larger flocs, and higher molecular weight for better 288 sedimentation into the pellet during centrifugation. PEG methods have been found to be more 289 commonly applied to surface water samples, while OF methods have been more commonly 290 used to concentrate wastewater samples (58). This tendency may be the result of laboratory 291 preferences or the fact that wastewater samples typically have higher viral titers and a loss of 292 viruses during acidification may be negligible and allow for adequate detection. The 293

inconsistency and lack of justification for method selection in many studies calls for a 294 systematic study to inform guidance and standardization with respect to the application of 295 concentration methods (3). 296

Fout and Cashdollar (2016) indicated that USEPA Method 1615 may be adapted for the 297 detection of adenoviruses (59). It is recommended to explore modifying existing methods with 298 secondary concentration by PEG precipitation to uphold virus recovery and minimize 299 inactivation during sample processing and detection by culture-based methods. Additionally, 300 PEG and OF methods should be compared for other viruses of interest to determine if there are 301 significantly different performance characteristics. Standard methods for the detection of enteric viruses in the environment may require customization for virus families due to the 303 diversity in virus stability, and water type. There is not likely one method that would produce a 304 high recovery of all viruses and surrogates of interest as there is high phenotypic and 305 biochemical diversity among viruses and phages. For example, all enteric viruses and F-specific 306 phage are non-enveloped and considered to be more stable in the environment than enveloped 307 viruses (e.g. influenza, coronaviruses, hepatitis B) (28, 35). While this information may add 308 complexity to existing multi-step procedures, this guidance will benefit the quality of 309 information that is gathered to inform risk assessments of recreational and drinking waters. 310

Appropriate process controls are necessary to provide an indication of the performance 311 of virus detection methods and provide a level of quantification with respect to recovery. 312

However, the recovery of a PCV does not provide an understanding of the mechanisms that 313 contributed to virus losses such as inactivation, poor de-adsorption during elution, or poor 314 flocculation unless the PCV has properties similar to the target virus. MS2 has been proposed 315 and used in numerous studies as a PCV (5, 60-64). However, the results of this study suggest 316 that the use of MS2, and other surrogates that may be unstable during sample processing, 317 should be done with caution or avoided in the case of acidification protocols when culture-318 based detection is employed. Alternatively, murine norovirus (MNV1) has been suggested as a 319 PCV (9, 22) and it has been found to be stable across the pH range from 2 to 10 (27). 320 A limitation of PEG methods is that the chemical conditions may contribute to the 321 inactivation of enveloped viruses by disrupting the lipid bilayer during concentration as has 322 been seen for influenza and murine hepatitis virus (+ssRNA, coronavirus), which may be 323 attributed to the NaCl concentrations (28). The selection of a secondary concentration method may be affected by the limitations of the detection method. For example, where PCR detection 325 methods are employed (e.g. for the detection of noroviruses which do not have established 326 culture-based methods), the inactivation of viruses may not be a primary concern so long as no 327 damage is caused to the genome during sample processing. However, secondary concentration 328 methods that have improved virus recovery and reduced co-concentration of PCR-inhibitors 329 would be beneficial for PCR detection methods. A perceived limitation of PEG may be the risk. An overnight incubation step still allows for these methods to provide results in a 1-day 337 turnaround when coupled with qPCR. This study and several others have completed PEG 338 methods with acceptable results with an incubation time of 1h. 339

In conclusion, this study presents primary data indicating the inactivation of coliphage 340 by OF acidification during secondary concentration of VIRADEL detection methods; though the 341 results from this study are applicable to elutions from ultrafiltration as well. Acidification 342 methods for the concentration of viruses should only be employed where viral stability at low 343 pH conditions has been confirmed or when only molecular based assays are used for virus 344 detection. Other alternative methods, such as PEG precipitation, are available that can produce comparable or improved recoveries during concentration procedures that substantially 346 minimize the inactivation of enteric viruses for culture-based detection. 347 from 10 1 to 10 8 copies of plasmid/well. It was assumed that one copy number was the 400 equivalent of one enteric virus unit. The efficiency of the qPCR assay was 101.5% +/-5% with R 2 401 values of 0.98-1.00, and slope between -3.21 and -3.50. 402

PCR inhibition was determined by spiking a concentrated HAdV41 DNA stock (6.4x10 4 404 gc/µl) into twofold serial dilutions (no dilution, 1:2, 1:4, 1:8) of a nucleic acid extract of each 405 water type as well as into reagent-grade water (Milli-Q, Water Purification System, Millipore, 406 Ontario) to serve as a positive control and subject to PCR as described above. The PCR 407 inhibition test determined that the following dilutions of samples minimized inhibition of the 408 HAdV41 qPCR assay by <1 Ct when compared to the positive control for the following water 409 types: 1:2 for all groundwater samples; 1:4 for the prepared and spiked surface water samples; 1:8 for prepared and spiked wastewater samples; and 1:2 for all suspended pellet samples. 411

These dilutions were used for all respective HAdV-qPCR reactions. 412

One litre samples of each of the following water types were prepared: wastewater 414 (WW; secondary effluent), concentrated groundwater (GW; from 1000 L), and concentrated 415 surface water (SW; from 80 L). Bulk concentrated groundwater and surface water (river water) 416 this study and would not bias the study outcomes with respect to evaluating OF or PEG 431 flocculation. Three flocculation methods were performed in duplicate; one acidification method directed away or opposite from the pellet location. The pellet was suspended in about 6 ml (± 1 455 ml) of PBS. MS2 and HAdV41 were enumerated in the suspended pellet as described above. 456

The % recovery of MS2 and HAdV41 following secondary concentration in the 458 suspended pellet and supernatant was calculated for each test using the initial spiked 459 concentration as the baseline. The remaining fraction of virus was considered "unaccounted". 

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FIG 1 Flocculated suspensions of surface water. Duplicate suspensions following OF lacking 709 visible flock (A)

HAdV41 enumerated by cell 717 culture in percent infectious units (IU) (B), and HAdV41 enumerated by qPCR in percent gene 718 copies (%) (C), by three concentration methods (PEG with 1.5 or 0.5 M NaCl and organic 719 flocculation [OF]) performed on wastewater (WW, 1L) and BE NanoCeram ® elution from 720 groundwater (GW; 1000 L) and surface water (SW, 80 L)

FIG 4 Virus adsorption and elution (VIRADEL) bench-top apparatus using NanoCeram ® positively 737 charged pleated cartridge filters. steps for acidification and PEG flocculation methods

by OF, and two methods using PEG with 0.5 and 1.5 M NaCl, respectively. Following flocculation 433 and prior to concentration by centrifugation, 10 ml samples were collected from each 150 ml 434 flocculation vessel to determine potential loss of infectivity and detection of viruses due to each 435 flocculation process. 436

Flocculation by acidification was performed according to the OF method described by 438 the USEPA (6). Spiked samples (150 ml each) were stirred using a magnetic stir bar and plate at 439 120 rpm to form a vortex and pH was adjusted to 3.5 ± 0.1 by adding 1M HCl. Samples were 440 stirred at 60 rpm for 30 min at room temperature (21 o C ± 1 o C) to facilitate floc formation (FIG ) . 441

Flocculation by PEG was performed according to (8)