Evaluation of Phi6 Persistence and Suitability as an Enveloped Virus

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Evaluation of Phi6 Persistence and Suitability as an Enveloped Virus Surrogate Nathalia Aquino de Carvalho, Elyse Stachler, Nicole Cimabue, and Kyle Bibby Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01296 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017

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Environmental Science & Technology

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Evaluation of Phi6 Persistence and Suitability as an

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Enveloped Virus Surrogate

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Nathalia Aquino de Carvalho1, Elyse N Stachler1, Nicole Cimabue1, and Kyle

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Bibby1,2*

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Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15261,

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USA.

Department of Civil and Environmental Engineering, and 2Department of

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*

Corresponding author:

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Mailing address: Department of Civil and Environmental Engineering, Benedum Hall

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3700 O’Hara Street, Pittsburgh, PA 15261. Phone: 412-624-9207; E-mail:

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[email protected]

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Keywords: Phi6, enveloped virus, virus, surrogate, persistence, Ebola

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Abstract

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Recent outbreaks involving enveloped viruses, such as Ebola virus, have raised

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questions regarding the persistence of enveloped viruses in the water environment.

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Efforts have been made to find enveloped virus surrogates due to challenges

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investigating viruses that require biosafety-level 3 or 4 handling. In this study, the

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enveloped bacteriophage Phi6 was evaluated as a surrogate for enveloped waterborne

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viruses. The persistence of Phi6 was tested in aqueous conditions chosen based on

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previously published viral persistence studies. Our results demonstrated that the

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predicted T90 (time for 90% inactivation) of Phi6 under the 12 evaluated conditions

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varied from 24 minutes to 117 days depending on temperature, biological activity, and

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aqueous media composition. Phi6 persistence was then compared with persistence

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values from other enveloped viruses reported in the literature. The apparent suitability of

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Phi6 as an enveloped virus surrogate was dependent on the temperature and

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composition of the media tested. Of evaluated viruses, 33%, including all conditions

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considered, had T90 values greater than the 95% confidence interval for Phi6.

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Ultimately, these results highlight the variability of enveloped virus persistence in the

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environment and the value of working with the virus of interest for environmental

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persistence studies.

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1. INTRODUCTION

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Recent disease outbreaks of enveloped viruses, such as Ebola virus,1,

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2

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Coronaviruses (e.g. MERS and SARS),3-5 Hantavirus,6 and Lassa virus,7, 8 have raised

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questions regarding the appropriate management of infectious patient waste, including

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wastewater, and the persistence of these viruses in the water environment.9 Multiple

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enveloped viruses require biosafety level (BSL) 3 or 4 handling, such as Ebola virus,

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Marburg virus, Hantavirus, Lassa virus, and SARS and MERS Coronaviruses.10 It is

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challenging to perform environmental persistence studies with these pathogens

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because access to BSL3 and BSL4 laboratories is expensive and limited. Historically it

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has been assumed that most enveloped viruses are rapidly inactivated in the

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environment;11,

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example, infectious Ebola virus persisted in sterile wastewater for at least eight days.13

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In addition, a recent review highlighted that the T90 (time for 90% virus inactivation)

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varied from hours to months for enveloped viruses under relevant environmental

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conditions.11 Consequently, it is desirable to work with agents that do not require access

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to BSL3 or BSL4 laboratories to conduct environmental persistence studies of these

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viruses. Subsequently, efforts have been made to find appropriate virus surrogates12, 14-

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17

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within lower biosafety levels.

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however, recent studies have undermined this assumption. For

– viruses that mimic the behavior of the pathogen of concern but can be evaluated

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Bacteriophage Phi6 is a candidate surrogate for enveloped viruses. Phi6 is a

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member of the family Cystoviridae and is among the few bacteriophages that have a

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lipid envelope.18 This bacteriophage has a double-stranded RNA genome of 13.4 kbp,19

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a diameter of 75 nm,20 a nucleoprotein capsid of 60 nm in diameter,20, 21 and 10 major 3 ACS Paragon Plus Environment


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coat proteins.21, 22 Phi6 has been widely used as an enveloped virus surrogate12, 14, 16, 17,

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23-26

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Pseudomonas syringae,27 are nonpathogenic to humans and require minimal

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containment (BSL 1).26 Second, its envelope makes it a potentially suitable model for

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studies of mammalian enveloped viruses due to physiological similarity.28 Third, Phi6

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and its host are easily cultivated in the laboratory, the analysis time is relatively rapid,

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and the assays are cost-effective.14 Other bacteriophage members of the Cystoviridae

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family are also enveloped and share similar characteristics;27, 29, 30 however, Phi6 was

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the first to be isolated18 and is the best characterized,31-33 which is why it has historically

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been chosen as an enveloped virus surrogate.

and offers many advantages as a potential surrogate. First, both Phi6 and its host,

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Despite the aforementioned advantages as an enveloped virus surrogate, the

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suitability of Phi6 for this application has yet to be formally evaluated across a wide

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range of conditions in liquid media. The appropriate surrogate choice should have a

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similar inactivation profile to the virus of comparison. The inactivation behavior for

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persistence studies is commonly represented by the time necessary for 90% reduction

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of the virus titer (T90), which assumes linear inactivation. Consequently, the evaluation

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of the potential surrogate can be performed by comparing its T90 value with the one of

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the pathogen of concern.

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In this study, the bacteriophage Phi6 was evaluated as a surrogate for enveloped

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virus persistence in liquid. Application of Phi6 to study enveloped virus transport or

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persistence on surfaces was not considered. The persistence of Phi6 was evaluated in

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representative aqueous conditions chosen based on previously published studies

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investigating the persistence of pathogenic enveloped viruses in liquid media. 4 ACS Paragon Plus Environment

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Additionally, a regression model for the effect of temperature on the persistence of Phi6

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in DI water was developed. Finally, to assess the suitability of Phi6 as a surrogate for

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enveloped viruses, we performed a meta-analysis to compare the persistence of Phi6

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with other enveloped viruses reported in the literature.

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MATERIALS AND METHODS

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Phi6 stock preparation and quantification

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Bacteriophage Phi6 and its host Pseudomonas syringae (HB10Y) were kindly

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provided by Dr. Leonard Mindich (Public Health Research Institute Center, New Jersey

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Medical School - Rutgers, The State University of New Jersey). Pseudomonas syringae

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was propagated in 6 mL of Luria-Bertani (LB) Broth Lennox (Fisher Scientific)

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containing 5 g L-1 NaCl and incubated overnight at 25°C. Phi6 bacteriophage was grown

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in Pseudomonas syringae by overlaying 35mL of LB agar (mixture of LB and Agar

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Bacteriological (Agar no.1 – OXOID) to reach 1.5% agar) with 6 mL of diluted soft agar

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(3 mL LB broth plus 3 mL LB soft agar – 0.6% agar) containing 1 mL of an overnight

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culture of HB10Y and 105 PFU Phi6. The plate was incubated overnight at 25°C. The

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top layer was harvested and purified by centrifuging at 14,000 rpm for 15 minutes at

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4°C. The supernatant Phi6 stock was enumerated and stored at -80°C. This

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propagation method of the bacteriophage Phi6 was based on a previously described

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procedure,34 and similar variations of the method were used in published studies.17, 24, 35

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Media Preparation



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Experiments were conducted in cell culture media (Dulbecco’s Modified Eagle’s

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Medium, D5030 – Sigma) and guinea pig sera (Sigma) as previously described by

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Piercy et al.36 DI water was produced by a Millipore Synergy® UV Water Purification

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System and stored at room temperature. Tap water was collected from the faucet in the

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Environmental Engineering laboratory at the University of Pittsburgh on 04/01/2015.

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The water authority servicing this building is the Pittsburgh Water and Sewer Authority,

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which uses the Allegheny River in Pittsburgh as the source water. Typical water quality

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parameters for this water are pH = 7.6, conductivity = 300 µS/cm. The tap was run for 5

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minutes to purge the system and samples were collected in sterile 50 mL Falcon tubes.

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The free chlorine of the tap water was measured using a Hach kit (DR900, program 80)

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and dechlorinated using a 46 g/L solution of sodium thiosulfate.37 The free chlorine was

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measured again after sodium thiosulfate addition to ensure dechlorination of the tap

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water. River water samples were collected from the Allegheny River in Pittsburgh on

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02/04/2016, sealed in sterile plastic bottles and transported on ice to the University of

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Pittsburgh. Two bottles were immediately stored at 4°C and two were autoclaved before

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storage at 4°C. Experiments began the same day, and remaining samples were stored

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at -80°C for future characterization. Both autoclaved and non-autoclaved samples were

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sent to Microbac Laboratories (Marietta, OH) overnight on ice for physical and chemical

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analysis (SI Table S1). Wastewater influent was collected from an anonymous regional

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wastewater treatment plant in western Pennsylvania that serves a population of

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approximately 60,000 from seven communities. Samples were collected in sterilized

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bottles and kept on ice while transported to the lab. Upon arrival to the lab, samples

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were frozen at -80°C to minimize changes in the chemical composition. On the day of


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the experiment, a sample bottle was thawed and autoclaved for 15 minutes. The

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autoclaved sample was sent to Microbac Laboratories (Marietta, OH) for chemical

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analysis (SI Table S1).

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Phi6 persistence experiments

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A volume of 24 µL of Phi6 stock (Phi6 concentration equals 109-1011 PFU mL-1)

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was spiked into 20 mL samples of liquid media – cell culture media, guinea pig sera,

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deionized water (DI), dechlorinated tap water, autoclaved river water, river water, or

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autoclaved wastewater influent – to a final concentration of 106 – 108 PFU mL-1. Spiked

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samples were immediately mixed and separated into 30 individual aliquots of 0.5 mL,

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which were stored in the dark for persistence experiments: cell culture media at 4°C and

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room temperature (RT = 22°C ± 1°C), guinea pig sera at 4°C and RT, autoclaved river

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water and river water at 23°C, autoclaved wastewater at RT, dechlorinated tap water at

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RT, and DI at 4°C, 25°C, 37°C, and 45°C. At each sampling time point, three aliquots

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were selected from the test condition, and three technical replicates were measured for

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each of the aliquots. The double agar overlay plaque assay was used to determine the

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concentration of infectious particles of Phi6: a mixture of 2.5 mL of soft agar (0.6%

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agar), 200 µL of the host bacteria (Pseudomonas syringae), and 100 µL of the spiked

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water sample was plated over LB agar plates (1.5% agar). Dulbecco's Modified Eagle's

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medium (DMEM) was used for the dilution of samples for Phi6 quantification. Negative

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controls were DMEM without Phi6. For river water samples, both autoclaved and non-

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autoclaved, a filtration step (0.45 µm polypropylene syringe filters with PVDF

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membrane, ThermoFisher Scientific) was added before the plaque assay procedure at

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each time point in order to avoid the growth of endogenous bacteria that could interfere 7 ACS Paragon Plus Environment


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with the experiment. Plates containing between 10 and 100 plaques were counted after

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24 hours incubation at 25°C. Phi6 concentration from sample and technical triplicates

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was expressed in PFU mL-1.

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Data analysis

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Phi6 concentration at each time point was expressed as log10(Ct/C0) – where Ct

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is the virus concentration in PFU mL-1 at time t, and C0 is the mean virus concentration

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in PFU mL-1 at time zero. The theoretical lower detection limit for virus concentration in

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the experiment was 100 PFU mL-1 – a minimum of 10 plaques were counted, and the

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volume of original sample following dilution added to the plate was 0.1 mL. Undetected

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virus observations were assumed to have a concentration of 50 PFU mL-1 – half of the

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lower theoretical detection limit – for analysis. We ceased measuring Phi6 concentration

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when the total experiment duration was over or when the virus concentration values

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were under the detection limit for two consecutive sampling points. For the experiment

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performed at 45°C, we stopped sampling after two hours based upon preliminary

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experiments.

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Linear regression models for Phi6 inactivation were obtained using GraphPad

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Prism version 7.00 (La Jolla, CA), and used to calculate T90 values. GraphPad Prism

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was used to compare the slopes of different conditions by performing the extra-sum-of-

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squares F test. To assess data deviation from linearity, the Runs test was performed.

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Statistical comparison between T90 values was achieved using the 2-sample t-test with

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unequal variances from Minitab 17.3.1. The significance level was 95%, i.e. P values

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smaller than 0.05 were considered statistically significant.


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Literature Meta-Analysis

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A literature review was performed to obtain T90 values of enveloped viruses or

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regression models of enveloped virus persistence in aqueous media. The articles of

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interest were found by searching the database Compendex using the terms “virus” AND

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“persistence” in “all fields”. Additionally, references from the papers selected were

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checked and articles with relevant information were included. When T90 values were not

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explicit by the authors, they were read from graphs or calculated from linear regression

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models.

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Published studies of viral persistence were performed in different pH,

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temperature, and salinity conditions; therefore, to enable comparison between viral

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persistence, we chose to include the values of viral persistence in a pH range of 6 – 9,

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and 0 ppt salinity, when specified. This pH range was chosen because most organisms

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present greatest stability at a near-neutral pH,38 and the salinity constraint was chosen

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to eliminate the effect of salinity in viral inactivation.39, 40

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RESULTS

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Phi6 persistence

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The persistence of bacteriophage Phi6 was calculated from each experimental

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condition: cell culture media (4°C and RT), guinea pig sera (4°C and RT), river water

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(autoclaved and non-autoclaved at 23°C), autoclaved untreated wastewater (RT),

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dechlorinated tap water (RT), and deionized water (4°C, 25°C, 37°C, 45°C) (Table 1).



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Phi6 inactivation followed first-order kinetics in all tested conditions – i.e. none of

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the linear regression models had a statistically significant deviation from linearity. Two

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alternative non-linear regression models were tested as a possible better fit for the

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persistence curves. When compared to the linear regression model, the one-phase

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exponential decay curve was the best fit for non-autoclaved river water, and the second

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order polynomial (quadratic) was the best fit for dechlorinated tap water, guinea pig sera

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at RT, and DI water at 4°C and 45°C (SI Table S2). Non-linear models have been

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previously reported for Phi6 inactivation in pasteurized sewage.17 Published studies of

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enveloped virus persistence have reported T90 values calculated from linear regression

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models; therefore, we utilized linear regression models for all conditions as the linear

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regression models were statistically valid and enabled the comparison of T90 values

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obtained in this study with literature values. Viral persistence significantly decreased

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over time (P < 0.05; i.e. the slopes were significantly different from zero) for all

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conditions except for guinea pig sera at 4°C (P = 0.5561) and DI water at 4°C (P =

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0.0878).

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Inactivation of Phi6 was assessed for different liquid media under the same

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temperature conditions to evaluate the role of aqueous media composition on Phi6

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persistence. Phi6 persistence in cell culture media and guinea pig sera was measured

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over a period of 46 days, and in DI water and autoclaved wastewater over a period of

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28 days. There was a statistically significant effect of matrix composition on Phi6

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persistence when the experiment was conducted at RT (Figure 1.A, P < 0.0001), but it

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was not significant at 4°C (Figure 1.B, P = 0.8905). In the experiment at RT, Phi6


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inactivation was fastest in DI, followed by autoclaved wastewater, guinea pig sera, and

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cell culture media.

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Phi6 persistence in autoclaved and non-autoclaved river water was measured

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over 28 days at 23°C (Figure 1.C) to investigate the role of microbiological activity on

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Phi6 inactivation. Phi6 inactivation was significantly faster in non-autoclaved river water

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when compared to autoclaved river water (P < 0.0001).

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To assess the role of temperature on inactivation rate, Phi6 persistence was

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quantified in DI water at 4°C, 25°C, 37°C, and 45°C (Figure 1.D). DI water was chosen

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for this evaluation to eliminate the confounding effect of media composition and

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biological activity. The effect of temperature on Phi6 persistence was statistically

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significant (P < 0.0001), with Phi6 persistence decreasing with increasing temperature.

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At 45°C, Phi6 abundance decreased to below the limit of detection within 2 hours. The

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temperature effect was also observed in other matrices considered: the inactivation

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kinetics of Phi6 were significantly faster in guinea pig sera (P < 0.0001) and cell culture

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media (P = 0.0003) at RT when compared to 4°C.

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The effect of temperature on viral inactivation has previously been reported in the

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literature.39-44 We subsequently developed a temperature model for Phi6 persistence in

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DI water (Figure 2). DI water was chosen to avoid the interference of biological activity

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and media composition. The relationship between log10(T90), where T90 is in days, and

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temperature (°C), followed the linear regression model log10(T90) = 2.23 – 0.082 *

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temperature (R2 = 0.97) (Figure 2).



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Figure 3 is a compilation of T90 values obtained from the Phi6 persistence studies

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and T90 values of enveloped viruses from the literature. Overall, the persistence of

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enveloped viruses varies widely for comparable conditions, and is highly dependent

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upon virus type, aqueous matrix composition, and temperature. For our meta-analysis,

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we considered room temperature to be from 20°C to 25°C. Based on our temperature

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model for Phi6 persistence in DI water, the difference in T90 values between

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temperatures of 20°C and 25°C would be 2.4 days.

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Comparing Phi6 T90 values from our study with T90 values of other enveloped

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viruses published in the literature, we found that Phi6 inactivation was more rapid, i.e.

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reported viral T90 values were above the confidence interval for Phi6, than all other

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viruses in guinea pig sera at RT (2 viruses), and wastewater at RT (4 viruses).

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Conversely, all viruses considered in cell culture media at 4°C (7 viruses), DI at 4°C (28

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viruses), dechlorinated tap water at RT (3 viruses), and guinea pig sera at 4°C (2

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viruses) had mean T90 values under the upper bound of the 95% CI for Phi6. The

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percentages of the viruses with greater persistence than Phi6 were 67% (6/9) for natural

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waters at RT, 11% (2/18) for cell culture media at RT, 84% (16/19) for DI at RT, and

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50% (2/4) for sterilized wastewater at RT.

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In order to expand our evaluation of Phi6 suitability as a surrogate for enveloped

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viruses, we compared Phi6 T90 values obtained in the literature with T90 values of other

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enveloped viruses from previously published studies. We considered both the suitability

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of Phi6 as a conservative enveloped virus surrogate that should persist longer than

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previously tested enveloped viruses that have been tested and, more specifically, the

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suitability of Phi6 as a surrogate of specific enveloped viral pathogens. 12 ACS Paragon Plus Environment

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As our study and one prior published study overlapped in a single evaluated

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condition (sterilized wastewater at RT), we used a 2-sample t-test with unequal

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variances to compare our Phi6 T90 value with that reported by Ye et al. (2016).12 The

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verification of variance inequality was performed with the F-test (P < 0.001), and the two

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T90 values were not statistically significantly different (P = 0.256). The percentages of

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the viruses investigated that were more persistent than reported Phi6 T90 values from

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other studies were 33% (22/67) for DI at 17°C, 33% (21/63) for DI at 27-28°C, 100%

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(1/1) in wastewater at 10°C, and 0% (0/1) for sterilized wastewater at 10°C.

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Accounting for all enveloped viruses obtained from the meta-analysis, 33%

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(76/228) had mean T90 values higher than the upper limit of the confidence interval of

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Phi6. Considering viruses that had data available for at least three conditions, only

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strains of influenza virus (H2N4, H5N7, H9N2, H11N6, and H12N5) never had a T90

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value higher than the upper limit of the confidence interval of Phi6.

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DISCUSSION

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We assessed Phi6 persistence under representative aqueous conditions

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selected from the literature and analyzed the suitability of Phi6 as a surrogate for

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enveloped virus persistence. We found that Phi6 inactivation was significantly affected

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by temperature, biological activity, and liquid media composition, consistent with

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previous reports for enveloped viruses. In addition, we found that enveloped virus

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persistence varies widely and the apparent suitability of Phi6 as a surrogate is

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dependent upon the evaluated condition. 13 ACS Paragon Plus Environment


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Environmental factors affecting virus inactivation

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Our results demonstrated that temperature, biological activity, and matrix

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composition have a significant effect on the persistence of Phi6. These trends are

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consistent with those previously observed for other enveloped viruses.12,

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Moreover, our meta-analysis highlights that virus persistence in water also varies

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between viral strains.11, 49, 55

15, 36, 39-54

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In the present study, Phi6 inactivation in all evaluated conditions followed first-

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order kinetics, consistent with previous findings.12 In five cases, the best-fit regression

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model for Phi6 persistence was found to be a one-phase exponential decay or second

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order polynomial. This inactivation behavior suggests that a subset of the viruses may

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persist longer than predicted by a linear decay model. An apparent decrease in the Phi6

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inactivation rate was also observed in sewage in a previously published study.17 The

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mechanism that promotes such virus resistance behavior is not yet understood;

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however, attempts to model apparent biphasic pathogen decay are ongoing.56 It is

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critical to elucidate the mechanisms of enhanced persistence and to further consider the

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fraction of viral populations with potentially enhanced persistence in future studies

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evaluating viral persistence and disinfection.

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The effect of matrix composition on viral persistence may be due to multiple

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factors. Organic content can protect microorganisms from disinfection processes57 and

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can be used as a nutrient supply by active microorganisms, possibly supporting the

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growth of microbial predators.38 In addition, ammonia inactivates viruses by genome

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degradation, and RNA viruses have been shown to be more sensitive to inactivation by


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ammonia than DNA viruses.58 Aggregation or adsorption of viral particles to particles in

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liquid media has been previously suggested to play a role in rapid virus decrease, which

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would be only an apparent viral decrease and not true virus inactivation.13 Although we

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found a statistically significant difference (P < 0.0001) between Phi6 inactivation in

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guinea pig sera and cell culture media, a previous study investigating Ebola virus and

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Marburg virus in the same experimental conditions found no significant difference in

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virus survival.36 This variation suggests that inactivation mechanisms related to the

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matrix composition are different between enveloped virus types. Moreover, a statistically

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significant difference (P < 0.0001) between Phi6 persistence in DI water and

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dechlorinated tap water at room temperature was observed, confirming that aqueous

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media composition has a significant effect on virus persistence even when comparing

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two fairly clean matrices.

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The role of temperature in viral persistence as observed in this study has been

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widely reported. Decreasing persistence with increasing temperature is attributed to the

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denaturation of proteins and nucleic acids, and the increased activity of extracellular

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enzymes.38, 43 The difference in inactivation rates between matrices for the most rapid

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(DI water) and least rapid (cell culture media) Phi6 decay time at room temperature is

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smaller than the difference in inactivation rates between the lowest (4°C) and highest

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(45°C) temperatures in deionized water; therefore, we observe that the temperature

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effect is more significant than the matrix composition effect. Consistent with this

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observation, it has been previously reported that temperature, organic matter, and

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aerobic microorganisms can influence virus survival in water, and that temperature has

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the most critical impact on virus persistence.43 15 ACS Paragon Plus Environment


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The effect of biological activity on viral persistence has been previously studied,

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and is suggested to be due to microbial predation59 or cleavage of viral proteins by

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proteolytic bacterial enzymes.60 Biomacromolecules that result from microbial activity

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such as proteases, deoxyribonucleases (DNase), and ribonucleases (RNase) have

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been reported to have a virucidal effect.61 Non-specific proteases and DNase/RNases

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resulting from microbiological activity are proposed to inactivate virus by catalyzing viral

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protein envelope (capsid) decay and disintegration of the nucleic acid core (DNA/RNA)

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of viruses.61 The specific mechanisms for inactivation of enveloped viruses due to

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biological activity are not well understood, and this is a necessary area of further

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investigation. This effect is associated with sterilization practices during laboratory

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experiments and is often necessary for subsequent viral culture. Sterilization was

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observed to enhance Phi6 persistence in liquid media for this and previous studies;

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however, previous work with murine hepatitis virus (MHV) demonstrated similar decay

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rates between pasteurized and non-pasteurized wastewater at 25°C,12 suggesting that

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this trend may not be universal. Based upon these observations, it is recommended that

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virus persistence studies are performed in non-sterilized media, when possible, or that

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authors note this likely enhanced persistence due to sterilization.

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Suitability of Phi6 as a surrogate for enveloped viruses

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Consistent with a recent review,11 our meta-analysis suggests that enveloped

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virus persistence varies by orders of magnitude depending on the environmental

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conditions, and it may take hours to weeks for a one log10 decay of various enveloped

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viruses under relevant environmental conditions. This finding is contrary to the common

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assumption that enveloped viruses are rapidly inactivated in the environment. The 16 ACS Paragon Plus Environment

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persistence of enveloped viruses in liquid may be of potential concern for secondary

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disease transmission, especially during wastewater handling in outbreak response.9

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This study highlights the continued importance of environmental persistence studies for

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emerging enveloped viruses.

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For Phi6 to be a desirable surrogate, the T90 value of Phi6 should be similar

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(within the 95% CI) or higher than the T90 of the virus of interest. The apparent suitability

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of Phi6 as a surrogate was dependent on the evaluated condition. All evaluated viruses

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with at least three available conditions had a T90 value above the 95% confidence

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interval for Phi6 under at least one condition, with the exception of some strains of

364

influenza virus. Subsequently, Phi6 cannot be considered to be a universally

365

conservative surrogate for enveloped viruses in aqueous media. These results also

366

highlight inter-virus variability in persistence trends under different conditions. To

367

overcome this limitation, it would be necessary to identify other enveloped viruses that

368

could be used as surrogates, or to work with the actual pathogen of interest. In general,

369

physiological differences between the chosen surrogate and pathogen of interest may

370

play a role in altering virus persistence. For instance, Phi6 has a dsRNA genome,

371

making it unique from many human enveloped viruses, and providing a potential

372

explanation for the observed differential persistence between Phi6 and enveloped viral

373

pathogens. Moreover, we indicated that Phi6 was an adequate surrogate when its

374

inactivation was significantly slower than the virus of concern; however, this assumption

375

may lead to overestimating virus survival and unnecessary public safety policies. If

376

surrogate work is necessary, it would be best to identify surrogates that mimic viral



Page 18 of 34

377

pathogen persistence as closely as possible, and to include multiple surrogates to

378

capture the inherent variability of environmental viral persistence.

379

Study limitations

380

This study had three main limitations. First, our analysis of Phi6 suitability as an

381

enveloped virus surrogate was limited by published data. In addition, many studies do

382

not report standard deviations, limiting our ability to statistically compare these values

383

with observed Phi6 persistence. Second, in cases where T90 values or regression

384

models for virus inactivation were not reported in the study, T90 values were estimated

385

from graphs presented. Third, virus inactivation has been shown to be affected by pH

386

and salinity;39, 40, 62 however, these factors were not considered in the current study.

387

Implications

388

While methods are being developed to estimate viral persistence a priori (e.g.

389

censored regression modeling),63 the inherently high variability of viral persistence

390

necessitates pathogen survival studies to predict exposure after virus release in the

391

environment.11,

392

enveloped viruses; therefore, it is recommended that multiple surrogates are considered

393

to cover the wide range of virus persistence variability, and, when possible, the virus of

394

interest itself should be used to determine viral persistence. Consequently, appropriate

395

virus control and public health safety strategies can be developed for future cases of

396

enveloped virus outbreaks.

38

Our study demonstrates that Phi6 is not a universal surrogate for

397


Page 19 of 34

398


ASSOCIATED CONTENT

399

Supporting Information. Liquid media characterization (autoclaved wastewater,

400

autoclaved and non-autoclaved river water); Non-linear regression models for survival

401

of bacteriophage Phi6 in liquid media at different conditions; Detailed conditions and

402

time for 90% reduction of enveloped viruses infectivity (T90) from persistence studies

403

reported in the literature.

404

AUTHOR INFORMATION

405

Corresponding Author

406

*Phone: +1 (412) 624-9207; e-mail: [email protected]

407

Notes

408

The authors declare no competing financial interest.

409

ACKNOWLEDGEMENTS

410

This study was supported by the National Science Foundation grant CBET-8508415.

411 412

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TABLES

638

Table 1. Linear regression models for survival of bacteriophage Phi6 in liquid media at

639

different conditions.

Matrix

Temperature (oC)

T90 (d) (95%CI) A

Linear regression model B

First order rate constant k (d-1) (avg±SE)

Autoclaved river water

23

7.1 (5.8 to 9.3)

Y = -0.140*X

0.140 ± 0.014

Nonautoclaved river water

23

3.1 (2.3 to 4.7)

Y = -0.322*X

0.322 ± 0.046

Cell culture media

RT

8.4 (6.3 to 12.4)

Y = -0.119*X

0.119 ± 0.012

Cell culture media

4

116.9 (20.1 to +infinity)

Y = -0.009*X

0.009 ± 0.013

Dechlorinated tap water

RT

3.1 (2.2 to 5.3)

Y = -0.321*X

0.321 ± 0.048

Autoclaved wastewater influent

RT

2.5 (2.4 to 2.7)

Y = -0.398*X

0.398 ± 0.009

Guinea pig sera

RT

5.3 (5.0 to 5.6)

Y = -0.189*X

0.189 ± 0.003

Guinea pig sera

4

72.7 (46.5 to 166.5)

Y = -0.014*X

0.014 ± 0.002

DI water

4

66.1 (30.2 to +infinity)

Y = -0.015*X

0.015 ± 0.008

DI water

25

1.6 (1.4 to 1.8)

Y = -0.634*X

0.634 ± 0.030

DI water

37

0.34 (0.32 to 0.37)

Y = -2.907*X

2.907 ± 0.061

DI water

45

0.017 (0.015 to 0.021)

Y = -57.52*X

57.52 ± 2.26

640

A

T90 value: Time required for 90% reduction in virus infectivity.

641

B

Y = log10(C/Co); X = persistence in days. 30 ACS Paragon Plus Environment

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642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657

Figure 1. Experimentally determined Phi6 persistence and linear regression models of the bacteriophage Phi6 in liquid media under different conditions. A) Phi6 inactivation at room temperature (RT) in different matrices. The reduction of virus concentration was significantly different for the matrices considered (P < 0.0001). B) Phi6 inactivation at 4°C in different matrices. The reduction of virus concentration was not significantly different for the matrices considered (P = 0.8905). Viral persistence did not significantly decrease over time in guinea pig sera (P = 0.0861) and DI water (P = 0.5732). C) Phi6 inactivation at 25°C in sterilized and non-sterilized river water. Virus persistence was reduced significantly faster in non-sterilized river water than in sterilized river water (P < 0.0001). D) Phi6 inactivation in DI water. Viral persistence decreased with temperature increase (P < 0.0001). Triplicate measurements of triplicate samples were taken at each time point. Geometric symbols represent mean value of the log10 of the virus concentration at each sample time over the mean concentration at time zero. Error bars indicate 95% confidence intervals (n = 3). Error bars not evident on the figure are smaller than the geometric symbol. Grey line represents zero viral decay.

658



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659 660 661 662 663 664 665 666 667 668 669

Figure 2. Linear regression model for the change of Phi6 T90 value (days) with temperature variation. The relationship between log10(T90) and temperature was linear; therefore, increasing temperature causes time for 90% inactivation of the virus to decrease. Persistence studies were conducted in DI at four different temperatures (4°C, 25°C, 37°C, 45°C). Error bars indicate 95% confidence internals. Error bars not evident on the figure are smaller than the geometric symbol. The upper 95% CI of the log10 (T90) value at 4°C is large because viral reduction was not significant over time and the upper 95% CI of the slope was positive. For this experiment, the linear regression model obtained was Y = 2.23 – 0.082 * X, with R2 = 0.97 and decay rate (°C-1) (avg ± 95% CI) = 0.082 ± 0.046.

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Influenza Viruses NDV SARS Human Coronavirus Hantaan virus 100

Puumala virus Tula virus HIV Murine hepatitis virus Ebola FIPV CCHFV SFSV

10

TGEV

T90 (days)

IHNV Marburgvirus ADV CSFV BVDV BPIV-3 1

Feline Coronavirus Phi8 Phi6 current study Phi6 literature

0.1 RT NW

672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687

4

RT

CCM

4

17

RT 27-28 RT DI

DTW

4

RT GPS

10

RT

10

SWW

RT

WW

Temperature °C Matrix

Figure 3. T90 (days) of Phi6 obtained on this current study and of various enveloped viruses reported in the literature.12-15, 36, 39-43, 45-53, 55, 64-74 Details of persistence studies obtained from literature review are shown in Table S3. Error bars on “Phi6 current study” indicate 95% CI, and on “Phi6 literature” indicate standard deviation. RT = room temperature (20°C – 25°C); NW = natural water; CCM = cell culture media; DI = deionized water; DTW = dechlorinated tap water; GPS = guinea pig sera; SWW = sterilized wastewater; WW = wastewater. NDV = Newcastle disease virus; SARS = Severe acute respiratory syndrome; HIV = Human immunodeficiency virus; FIPV = Feline infectious peritonitis virus; CCHFV = Crimean-Congo hemorrhagic fever virus; SFSV = Sandfly fever Sicilian virus; TGEV = Transmissible gastroenteritis virus; IHNV = Infectious haematopoietic necrosis virus; ADV = Aujeszky's disease virus; CSFV = Classical swine fever virus; BVBD = Bovine viral diarrhea virus; BPIV-3 = Bovine parainfluenza virus type 3. HIV T90 (1 hour) in DTW was excluded from the graph for better visualization. 33 ACS Paragon Plus Environment


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Evaluation of Phi6 Persistence and Suitability as an Enveloped Virus

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