Inactivation Mechanisms of Human and Animal Rotaviruses by Solar

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Environmental Processes

Inactivation mechanisms of human and animal rotaviruses by solar UVA and visible light Elbashir Araud, Joanna L. Shisler, and Thanh H. Nguyen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06562 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Environmental Science & Technology

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Inactivation mechanisms of human and animal rotaviruses

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by solar UVA and visible light

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Elbashir Araud1, Joanna L. Shisler2, and Thanh H. Nguyen1

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Department of Civil and Environmental Engineering, University of Illinois at Urbana-

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Champaign, Urbana, Illinois, USA1; Department of Microbiology and Department of

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Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA2

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1 ACS Paragon Plus Environment


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ABSTRACT

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Two rotavirus (RV) strains (sialidase-resistant Wa and sialidase-sensitive OSU) were irradiated

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with simulated solar UVA and visible light in sensitizer-free phosphate buffered solution

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(lacking exogenous reactive oxygen species (ROS)) or secondary effluent wastewater (producing

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ROS). Although light attenuated for up to 15% through the secondary effluent wastewater

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(SEW), the inactivation efficacies increased by 0.7 log10 for Wa and 2 log10 for OSU compared

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to those in sensitizer-free phosphate buffered solution (PBS) after 4 hrs of irradiation. A binding

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assay using magnetic beads coated with porcine gastric mucin containing receptors for

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rotaviruses (PGM-MB) was developed to determine if inactivation influenced RV binding to its

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receptors. The linear correlation between the reduction in infectivity and the reduction in binding

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after irradiation in sensitizer-free solution suggests that the main mechanism of RV inactivation

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in the absence of exogenous ROS was due to damage to VP8*, the RV protein that binds to host

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cell receptors. For a given reduction in infectivity, greater damage in VP8* was observed with

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sialidase-resistant Wa compared to sialidase-sensitive OSU. The lack of correlation between the

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reduction in infectivity and the reduction in binding, in SEW, led us to include RNase treatment

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before the binding step to quantify virions with intact protein capsids and exclude virions that

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can bind to the receptors but have their capsid permeable after irradiation. This assay showed a

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linear correlation between the reduction in RV infectivity and RV-receptor interactions,

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suggesting that RV inactivation in SEW was due to compromised capsid proteins other than the

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VP8* protein. Thus, rotavirus inactivation by UVA and visible light irradiation depends on both

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the formation of ROS and the stability of viral proteins.

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INTRODUCTION

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Group A rotaviruses (RVs) are the major etiological agent of acute gastroenteritis in infants

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worldwide and accounted for 215,000 deaths in children in 2013.1 It has as low an infectious

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dose of fewer than 10 particles and is shed at high titers (1010 to 1012 particles per gram of stool)

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from infected persons.2-4 Despite the success of vaccines against RVs in the reduction of severe

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gastroenteritis among infants, RVs are still of major public health importance and the leading

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cause of diarrhea in children younger than five years old, globally.2, 5 RV outbreaks still occur

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worldwide due to the high genetic diversity of RVs and the lack of cross-protection.2, 5 There are

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eight groups of rotaviruses, referred to as A, B, C, D, E, F, G, and H. Humans are primarily

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infected by group A rotavirus, which can be further divided into different serotypes.6 The outer

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capsid glycoprotein (VP7) and the spike protein (VP4) differentiate RVs into 14 G

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(Glycoprotein) serotypes and 27 different P (Protease sensitive) genotypes.6 Currently, five

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serotypes (G1–G4, and G9) are the predominant circulating viruses, accounting for almost 95%

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of strains worldwide.7 In addition, reassortment of RV strains between animal and human has

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been reported.8-13 Thus, RVs are highly diverse, both antigenically and genetically.

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RV-contaminated water and food are the main source of RV outbreaks.14 In both developed and

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developing countries, infectious particles or genomes of human or animal RV are found in

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drinking water sources.15-19 The contamination of the drinking water source is attributed to

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inadequately treated wastewater.20 Also, it is troublesome that RV is detected on lettuce irrigated

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with surface water in the US.21 Thus, the risk of RV outbreaks may increase when contaminated

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surface water comes into contact with vegetables and seafood. To complicate matters, newly

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emerging RV strains due to intra-genogroup reassortment are reported constantly and may also

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add to current or future RV outbreaks.8, 22 Therefore, prevention of RV infection and outbreaks 3 ACS Paragon Plus Environment


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can be achieved by improving wastewater disinfection and by knowing the stability of RV in

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wastewater receiving surface water so that minimal contamination of food or drinking water

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sources can be planned.20, 23

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Solar inactivation of viruses is an important process that controls the water quality in a number

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of cases including point of use water treatment (solar disinfection; SODIS); waste stabilization

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ponds to treat municipal wastewater; reuse of treated wastewater for irrigation; discharge of

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treated wastewater to surface water bodies, which are also used for recreation.24-26 In solar

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disinfection, pathogen inactivation relies on solar irradiation, which directly or indirectly targets

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the microbes (endogenous) or causes reactions between the microbes and the reactive oxygen

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species (ROS) formed from natural sensitizers in water (exogenous).27, 28 Most solar disinfection

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studies use indicator bacteria and bacteriophages as surrogates for human enteric viruses.27,

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However, this is not a practical surrogate for RVs.29 Because there is strain-dependent

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susceptibility of RVs to solar disinfection (e.g., the porcine RV strain OSU is more resistant

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than the human RV strain Wa)30 and to disinfection by high pressure.31 These findings suggest

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that it is important to understand disinfection mechanisms of real pathogens instead of

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surrogates.

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Binding to host cell is the first essential step in virus infectivity. The RV VP4 spike protein

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attaches to host cell glycans, including gangliosides (N-acetylneuraminic acid (Neu5Ac) and N-

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glycolylneuraminic acid (Neu5Gc)) and histo-blood group antigens (HBGA). VP4 consists of

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two components VP8* and VP5. Located on the top of VP4, VP8* is essential for the early stage

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of the virus cell attachment, while VP5 is involved in the later stages of the cell membrane

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penetration and virus entry. Some RV strains (referred to as sialidase-sensitive) attach to sialic

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acid moieties on the surface of the cell. This attachment for sialidase-sensitive rotaviruses 4 ACS Paragon Plus Environment

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requires VP8* to mediate this interaction. Other RV strains, referred to as sialidase-resistant, use

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Neu5Ac and histo-blood antigen groups instead of sialic acid to bind to the host cells. Because of

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these two different binding mechanisms, these VP8* amino acid sequence is slightly different.

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Specifically, for VP8* binding to sialic acid domain, a common grove on the VP8* domain with

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two key amino acids at 157 and 187 position is important.32 In addition, amino acids at positions

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R101, Y189, and S190 are strictly conserved for rotavirus strains that are sialidase-sensitive. For

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some sialidase-resistant strains with VP4 genotype P4, P6, P8 and P11 the amino acid at R101,

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the key binding amino acid to sialic acid, is substituted with amino acids with hydrophobic side

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chains. This change disrupts the formation of the hydrogen bond and the interaction with the

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sialic acid. Replacing of amino acids residues Y155, Y188, and Y189 with other amino acid with

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hydrophobic side chains further precludes the binding to the sialic acid.33 However, knowledge

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on how RV capsid proteins may dictate resistance or susceptibility to inactivation is limited.

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To understand the stability of the RV capsid under solar irradiation, this study aimed to

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determine the mechanisms controlling inactivation of two RV strains representing the most

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predominant human and animal group A rotavirus genotypes. In addition to measuring the

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inactivation efficacy of these RV strains in solutions without exogenous ROS or in secondary

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effluent of wastewater (SEW) treatment, we also developed and calibrated binding assays

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combined with RNase treatment to determine the extent of damage to the RV capsid caused by

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solar UVA and visible light irradiation. Our findings demonstrated that design of UVA and

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visible light disinfection or prediction of rotavirus survival in solar UVA and visible light must

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consider both the formation of ROS and the stability of viral proteins.

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MATERIALS AND METHODS



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Rotavirus stock preparation. RV strains from two different serotypes were used in this study.

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The Wa strain (serotype G1P[8]) and OSU strain (serotype G5P[7]) were obtained from ATCC

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catalogue numbers VR2018TM and VR892TM, respectively. All strains were propagated in MA-

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104 cells cultured in Eagle's minimum essential medium (MEM) supplemented with 0.5 µg/ml

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trypsin (Sigma, St. Louis, MO), as previously described.34 RVs were purified and concentrated

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by sucrose cushion and centrifugation. Purified RVs were aliquoted and stored at -80 °C until

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used. RV plaque assays were conducted as described previously31, in which MA-104 cellular

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monolayers were used and plaques were visualized by using crystal violet staining.

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Sialic acid sensitivity assays. Rotavirus strains with different compositions of the binding

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proteins VP8* recognized different host cell receptors.35 To determine whether two strains of RV

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that differed in their mechanism of binding to the host cell receptors have different inactivation

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mechanisms, we selected two RV strains (OSU and Wa) that were likely to recognize different

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host cell receptors. To determine the sensitivity of the selected strains toward sialidase, we

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compared the titer of each strain after incubation with either untreated or NA-treated cellular

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monolayers as follows: MA104 cells in 6-well plates were washed twice with 2 mL of serum-

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free Minimum Essential Medium (MEM). Cellular monolayers were incubated in 1 mL MEM

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containing or lacking 40 mU/ml neuraminidase (Sigma, St. Louis, MO ) for 1 hr at 37 °C and 5%

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CO2. Next, monolayers were washed twice with 2 mL of cold MEM, and then ten-fold dilutions

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of viral solutions with initial titers of 2.5×106 and 1.0×107 PFU/mL for Wa and OSU strains,

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respectively, were inoculated onto different monolayers. Infected monolayers were incubated on

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ice for 60 min, with agitation every 10 to 15 min. Monolayers were washed twice with pre-

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warmed MEM medium to remove unattached viruses. Next, monolayers were overlaid with 2.5

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ml of 2X Eagle minimum essential medium (MEM) containing 2.5% agarose, Anti-Anti (10,000


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units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Gibco Amphotericin B)

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(Thermo Scientific, Rockford, IL), and 1µg/ml trypsin (Sigma, St. Louis, MO). These plates

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were incubated at 37 ˚C and 5% CO2 and examined for plaques at 3 days post-infection.

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Comparison of VP4 gene sequence of OSU and Wa strains. To confirm the differences on the

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sialic acid binding sites on the VP8* protein, the VP4 gene of OSU and Wa strains were

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amplified by RT-PCR and sequenced. Genomic RNA of OSU or Wa strain was extracted from

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140 µl of the virus stock using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA),

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according to the manufacturer’s manual. Based on GenBank published nucleotide sequences of

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OSU and Wa, two different pairs of primers for VP4 of OSU and Wa were designed and used in

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One-Step Reverse Transcription-PCR (RT-PCR) (Table 1S). The amplified VP4 band was

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visualized by 1% agarose gel electrophoresis and purified using QIAquick PCR Purification Kit,

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according to the manufacturer’s manual. To confirm identity of the studied strains, the purified

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VP4 was sequenced, using as set of primer walking (Table 1S), at the High-Throughput

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Sequencing and Genotyping Unit at the University of Illinois at Urbana-Champaign. Sequence

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conversion and alignment were conducted using BLAST (NCBI).

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Sampling and characterization of wastewater. Wastewater from secondary clarified effluent

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of the Northeast Wastewater Treatment Plant (NEP) in Urbana, Illinois was collected as

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previously described by Dong et al.36 This secondary effluent wastewater (SEW) was used to

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study exogenous inactivation of RVs triggered by UVA and visible light irradiation. The total

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organic carbon (TOC) of 16.5 mg/L, ammonia of 7.3 mg/L, and nitrate of 0.2 mg/L were

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detected.36 All experiments with SEW were conducted at its pH of 8.1. Absorbance of the SEW

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used for irradiation experiments is shown in Figure 4S.



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Solar UVA and visible light disinfection experiments. A sunlight simulator (Atlas Suntest(r)

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XLS+, photosimulator, Chicago, IL) with a xenon arc lamp was used for virus inactivation

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experiments. UVA/visible light with wavelength of 320 nm and above and intensity setting of

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400 W m−2 was used in all irradiation experiments. The light density and the wavelength cut of

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the sunlight simulator was measured using a StellarNet Inc. spectrometer. Because UVB was

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largely absent in the solar spectra collected in IL (Figure 1S and Table 2S) and the attenuation

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of UVB was at least twice more than UVA in water with comparable dissolved organic matter to

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wastewater using the correlations developed by Roberts et al.,37 solar UVA and visible light

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irradiation is more relevant. For this reason, we conducted the study with solar spectra without

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UVB. To confirm the removal of light under the UVA/visible (320 nm), Newport 320 nm cutoff

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filters (Atlas MTS, Cat.56052372) wer used. These filters were put on top of all reactors for solar

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disinfection and radical formation measurement. These reactors were made of 100-ml glass

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beakers, with edges covered with a dark tape to prevent the light penetration and reflection. The

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solution depth in the reactor was 1 cm. The irradiation experiments were conducted at 21 °C

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maintained by a circulated water bath. A magnetic stirrer was used for continuous stirring the

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solution in the reactors. The RV titer in each reactor was 105 PFU/ml. Titers were determined

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from aliquots withdrawn from the reactor at different time points. Dark controls were used in

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parallel with samples under the same conditions as a negative control, to confirm that the

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reduction of the virus titer was only due to the irradiation. At least three replicates were

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conducted with the same wastewater that was collected in one day. This wastewater was kept at

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4 ˚C in the dark and used for disinfection experiments within 2 weeks from the collection date.

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All the treated viruses were kept at -80 ˚C till all the plaque assays where conducted. The

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irradiation was monitored throughout the study. Experiments with two viruses were conducted


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either at the same time or one right after the other to reduce variability among experimental

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conditions. The initial titer in the inactivation solutions (Co) was determined using samples

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immediately after the viral stock was added and mixed with the inactivation solutions and right

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before irradiation. As shown in Figure 1S showing the spectra of the simulator and natural

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sunlight, 4 hrs irradiation by the simulator is equivalent to 4 hrs of sunlight at 1 PM in IL. A

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single peak at around 100 nm was observed using dynamic light scattering to indicate

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monodispersitiy of the viral suspension in SEW.

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Detection of oxygen reactive species (ROS) produced in wastewater. Phenol and Furfural

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alcohol (FFA) were used as probes to detect hydroxyl (˙OH) and singlet oxygen (1O2) radicals,

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respectively.31 Twenty milliliter of SEW samples were spiked with 100 µM of corresponding

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probes and irradiated under the same conditions as for UVA and visible light disinfection

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experiments (above) for 4 hrs. Samples of 400 µL were withdrawn every hour and the decay of

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different probes was detected by HPLC Reverse-phase Agilent series 1200 (Agilent

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Technologies) with Eclipse Plus C18 (3.5 µm) column was used for probes detection. The

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mobile phase was water and acetonitrile at a ratio of 50:50 for phenol and 40:60 for FFA, with

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flow rate of 0.3 ml/min and injection volume of 20 µl. The detection wavelength was 268 and

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216 nm for phenol and FFA, respectively. ROS measurement was conducted with SEW without

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spiked RV.

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PGM-MB binding assay. To determine the effect of UVA and visible light irradiation on RV

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binding to its receptor, assays were developed that discriminated between virions that retained or

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lost their ability to bind to their receptors. Porcine gastric mucin (PGM) was bound to magnetic

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beads (MB), as described previously for norovirus binding.38-40

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MagnaBind carboxyl-derivatized beads (Thermo Scientific, Rockford, IL) was washed three time 9 ACS Paragon Plus Environment

Briefly, one milliliter of


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with PBS buffer. A magnetic separation rack (New England Biolabs, Ipswich, MA) was used to

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collect beads after each wash. A ten milligram of type III mucin from porcine stomach supported

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by 0.5 to 1.5% sialic acid (Sigma, St. Louis, MO) was dissolved in one milliliter of conjugation

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buffer (0.1 M MES (2-(N-morpholino) ethanesulfonic acid), 0.9% NaCl, pH 4.7) and added to

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the washed MagnaBind carboxyl-derivatized beads. Then 100 µL of 10 mg/mL 1-ethyl-3-[3-

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dimethylaminopropyl] carbodimide hydrochloride (EDC) was immediately added as conjugation

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buffer to the beads and the mucin. The mixture was rotated for 30 min at room temperature at 8

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rpm. To remove excess mucin, beads were washed three times with PBS. A magnetic separation

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rack (New England Biolabs, Ipswich, MA) was used to collect beads after each wash. Finally,

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beads were re-suspended in 1 mL phosphate buffer solution (PBS) containing 0.005% sodium

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azide, and beads were stored at 4 °C till used.

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For each binding assay, 50 µL of each sample (with or without RNase treatment) and 70 µL of

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Porcine Gastric Mucin beads (PGM-MB) were added to a 1.5 mL low adhesion centrifuge tube.

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Sterilized PBS was added to this mixture for a total volume of 1 mL. This tube was gently

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shaken by a bench shaker at room temperature for 30 min at 8 rpm. Beads were washed three

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times with 1 mL PBS to remove unbounded particles. Beads were re-suspended in 140 µL of

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DNase/RNase-Free distilled water before being subjected to RNA extraction. To avoid detecting

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the noninfectious virus particles that had partially damaged capsid but still retained the ability to

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bind to the beads, RNase treatment was used for another set of samples. Specifically, 2 µL of

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RNase A/T1 (40 µg/ml RNase A and 100 U/ml RNase T) (Thermo Scientific, Rockford, IL) was

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added to 50 µL samples collected from the UVA and visible light disinfection experiments with

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wastewater and incubated at 37 °C for 30 min. After incubation with RNase, 2 µL of

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SuperRNase inhibitor (sigma-Aldrich) was added to the samples and incubated for 20 min at


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room temperature to inactivate the residual RNase. Then the RNase-treated samples were

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subjected to PGM-MB binding, RNA extraction, and RT-qPCR detection, as mentioned above.

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Note that RNase A degrades RNA at C, and U residues and RNase T1 degrades the RNA at G

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residue. The concentrations of RNase and RNase inhibitor were found through preliminary

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experiments. Specifically, different concentrations of the RNase mixtures (80, 60, and 40 µg/ml

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RNase A, and 200, 150, and 100 U/ml RNase T1) were added to the virus solution before the

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binding assay to determine the right concentration allowing degradation of naked RNA and the

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RNA inside the damaged capsid. At concentration higher than 40 µg/ml RNase A, and 100 U/ml

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RNase T1, no RNA was detected by RT-qPCR. From 10 PFU/mL to 107 PFU/mL of either OSU

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or Wa was used to establish the calibrations. This wide range was used to cover the detected

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infectivity needed to assess binding ability of rotavirus after irradiation.

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Quantification of RVs RNA by quantitative reverse transcription PCR (RT-qPCR). RT-

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qPCR amplification was used to detect and quantify RV genes after UVA/visible light irradiation

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or to calibrate the MBG-MB binding assays described above. The total RNA from untreated or

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UVA-visible light irradiated RVs was extracted using the RNeasy kit (Qiagen). This RNA was

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then reverse transcribed using the following sets of primers targeting the NSP3 genes of RV. For

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Wa and OSU, the forward primer JVKF (5'- CAG TGG TTG ATG CTG AAG AT -3') and

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reverse primer JVKR (5'- TCATTGTAATCATATTGAATACCCA -3'),41 were used to target

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amplicon of 120 bp. A PCR machine (Applied Biosystems, Foster City, CA) was used for RT-

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qPCR amplification. The targeted region of NSP3 was amplified using iTaq-Universal SYBR

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Green iScript reverse transcriptase (Invitrogen) according to the manufacturer’s protocol.

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Cycling parameters were as follows: 48 °C for 10 min for cDNA production, then inactivation of

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RT and activation of PCR enzymes at 95 °C for 1 min. For qPCR, there were 40 cycles of 94 °C



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for denaturation for 10 s, 30 s for annealing and extension at 50°C. Standard curves were

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generated by performing RT-qPCR reactions in parallel with samples containing a known

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concentration of the plasmid encoding the NSP3 gene. The PCR efficiency ranged from 90-99%

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with R2 of 0.97 to 0.99. Samples had a high enough concentration of RV that dilution 100 times

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was usually needed. This high dilution level avoided PCR inhibition.

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Effect of visible light and UVA irradiation on RV genome. To determine the effect of visible

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light and UVA irradiation on the RV genome, total RNA from 140 µL of purified Wa (initial

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titer of 2.5 x 106 PFU/mL) was extracted using RNeasy kit (Qiagen) and mixed with 800 µL of

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organic free phosphate buffered solution (PBS) containing 10 µl RNase-out (1:10

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dilution) (Sigma). A portion of 110 µL of RNA, from the 940 µL mixture in PBS solution was

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put in one of nine wells of a 96-well plate. The plate was covered by a Clear Adhesive Film (The

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Applied Biosystems® MicroAmp®) to prevent evaporation (Figure. 2S). The sides of the plate

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were also secured by regular tape to prevent evaporation. The control well was covered by dark

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tape to prevent irradiation exposure. A Newport 320 nm cutoff filter (Atlas MTS, Cat.56052372)

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was put on the top of the 96-well plate. The plate was irradiated in the sunlight simulator for 4

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hrs. The temperature was controlled around 21 °C during the irradiation by a circulated water

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bath. After 2 and 4 hrs of exposure, one well was covered by dark tape to stop the exposure to

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the light at that time point. The change in the amount of irradiated or the control RNA was

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quantified by two-step RT-qPCR (Applied Biosystems, Foster City, CA). VP4 segment of 2360

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bp was selected for the reverse transcriptase step. Reverse primers (VP4OSU 2340-2360) and

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(VP4Wa 2336-2358) (Table 3S) were used to produce cDNA in the size of 2,360 and 2,358 bp

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from VP4 of OSU and Wa, respectively. Ten microliters of irradiated and unirradiated RNA 12 ACS Paragon Plus Environment

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were used for first strand amplification (cDNA) using reverse transcriptase enzyme (BioLabs,

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New England). Ten microliters of the irradiated or unirradiated RNA samples were used for

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synthesizing cDNA using reverse transcriptase enzyme (BioLabs, New England). The denaturing

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step at 95 °C for 5 min was followed by two minute cooling on ice. Then 10 µL of reverse

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master mix contained the follows: 2µL of reverse primer (10 µM), 2 µL of M-MuL buffer, 1 µL

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of RNase-inhibitor, 1µL the RT enzyme, 1 µL of (10 µM) dNTP, and 3 µL of water were added

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to each denatured RNA. The RT step cycling was 5 min at 25 °C, 45 °C for 60 min, and 62 °C

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for 20 min, to inactivate the RT enzyme. For the qPCR step, 1.5 µL of the cDNA synthesized in

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the reverse transcription step was used with either primer pair VP4OSU 89-109 and VP4OSU

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173-195 or primer pair VP4Wa 202-225 and VP4Wa 119-140 to amplify a segment of 106 bp.

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Primers sequences are listed in Table 3S. Cycling parameters were as follows: 95 °C for 2 min,

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to activate the PCR enzyme. For qPCR, there were 40 cycles of 94°C for denaturation for 15 s,

274

and 50°C for 30 s for annealing and extension. General iTaq Universal SYBR Green and qPCR

275

kits from Applied Biosystems (Foster City, CA) were used for the qPCR step. The standard

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curves for the qPCR step were generated by performing qPCR on six aliquots of the cDNA with

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known concentration of the targeted fragment. Based on the standard curves, the qPCR

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efficiency was 115% for OSU and 112% for Wa. Coefficients of determination R2 were 0.99 in

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both cases.

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Statistical analysis. For each experimental condition and for each type of measurement (either

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inactivation or radical measurement), an experiment was performed at least three times

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independently. Data showing inactivation kinetics were presented as log10 of the negative value

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of the titer at a given irradiation time normalized by the titer at time zero. This parameter is

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referred to as log10 reduction in plaque forming unite (PFU). The reduction in binding was



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presented in terms of log10 of the negative value of the RT-qPCR results after binding assay for a

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given sample at a given irradiation time, normalized by the RT-qPCR results after binding assay

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for the sample collected at time zero. For all possible linear correlations between a pair of two

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variables, Pearson product-moment correlation analysis was conducted for all collected data. The

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significance of the linear correlation was based on the value of the Pearson’s correlation, p-value,

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and coefficient of determination R2.

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RESULTS AND DISCUSSION

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The effect of UVA and visible light irradiation on RV genome in sensitizer-free phosphate

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buffered solution. One potential reason for RV inactivation by UVA-visible light irradiation

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would be if visible light and UVA irradiation damaged the viral RNA, therefore preventing virus

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replication. To explore this possibility, RNA from RV strain Wa and OSU was extracted from

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untreated virions and then exposed to visible-UVA irradiation for 2 or 4 hrs. We exposed naked

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viral RNA instead of whole virions because naked RNA should be more susceptible to

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irradiation than RNA encapsidated inside the virions. Next, we conducted reverse transcription

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of the VP4 gene, a medium-sized segment of RVs genome. We chose this segment as a

300

representative of genomic damage that might occur in other segments of the RV genome. We

301

used a modified protocol that would PCR amplify undamaged RV RNA to cDNA genome

302

fragments in the size of 2360 bp for OSU and 2358 bp for Wa. We then quantified the presence

303

of cDNA by using qPCR, targeting a 106-bp region of the 2,360 or 2,358-bp amplicons. We

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tested 3 biological replication and 2 technical replicates for each irradiation condition. We found

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no statistically significant difference between samples irradiated for 2 hr or 4 hr versus the dark

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control (p=0.14 for OSU and p=0.29 for Wa). Thus, irradiation by solar UVA and visible light

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did not degrade the RV genome. 14 ACS Paragon Plus Environment

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Differences in the VP8* residues important for sialic acid interactions. The above data

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suggested that RV inactivation due to solar UVA and visible light irradiation most likely was due

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to capsid protein transformation and/or degradation. However, it is not known what RV capsid

312

proteins are damaged due to endogenous or exogenous reactive oxygen species (ROS). To

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answer this question, we examined the inactivation of two strains of RV that differ in their

314

mechanism of binding to the host cell receptors. We hypothesized that, if solar UVA and visible

315

light irradiation damaged these viral binding proteins, then RV would no longer be infectious.

316

Inactivation mechanisms of RV may be related to its ability to bind to the host cell surface. The

317

host cell receptors that are crucial for RV attachment and entry to the cell are well known 35. We

318

amplified and sequence the VP8* gene of two rotavirus strains Wa and OSU. When we aligned

319

the amino acid sequences of the VP8* gene of the rotavirus strains OSU and Wa, the amino acids

320

essential for sialic acid binding (101, 155, 188, and 190) were different, agreeing with reported

321

literature. In addition to comparing the amino acid sequence, we also experimentally determined

322

the sensitivity of these two strains toward sialidase by comparing the titer of each strain after

323

incubation with host cells with or without treatment with NA to remove sialic acid. As shown in

324

Table 1a, for the Wa strain, the titers of Wa RV propagated with cells not treated with NA were

325

from 2.9 to 3.4 fold higher than those propagated with cells treated with NA. This increase

326

suggested that the Wa strain is sialic acid-resistant because when sialic acid was removed, Wa

327

has better access to the host cell receptor (N-acetylgalactosamin). On the other hand, for the

328

OSU strain, the titers of RV infected cells treated with NA were from 0.2 to 0.3 fold smaller than

329

those of RV infected cells not treated with NA. This decrease suggests that when sialic acid was

330

removed from the host cells, OSU could not bind to the host cells and infect the cells, indicating



Page 16 of 32

331

that OSU is sialic acid-sensitive. We further tested whether OSU and Wa, whose most outer

332

binding protein VP8* has different amino acid composition (Table 1b) and binding mechanisms

333

to host cells, have different sensitivities toward solar UVA and visible light irradiation in the

334

presence or absence of reactive oxygen species (ROS).

335 336 337

Table 1a. Results of sialidase sensitivity assay of Wa and OSU strains Strain

Ratio of RV infected cells not treated p-value (-NA compared with with NA over RV infected cells treated +NA) with NA

OSU

0.2; 0.2; 0.3

4×10-5

Wa

2.9; 3.4; 3.0

0.03

338 339

P-values are from t-test comparing the titers of rotavirus infected cells treated or not treated with NA.

340

Table 1b. Alignment of VP8* portion of the VP4, that attach to sialic acid, for OSU and Wa RV strain

OSU 61 Wa 61

Amino acid positions

VEPLLDGPYQPTTFNPPTSYWVLLAPTVEGVIVQGTNNTDRWLATILIEPNVQTTNRIYN 120 VEPILDGPYQPTTFTPPNDYWILINSNTNGVVYESTNNSDFWTAVVAIEPHVNPVDRQYT 120

OSU 121 LFGQQVTLSVENTSQTQWKFIDVSTTTPTGSYTQHGPLFSTPKLYAVMKFSGRIYTYNGT 180 Wa 121 IFGESKQFNVSNDSN-KWKFLEMFRSSSQNEFYNRRTLTSDTRFVGILKYGGRVWTFHGE 179 OSU 181 TPNATTGYYSTTNYDTVNMTSFCDFYIIPRNQEEKCTEYINHGLPPIQNTRNVVPVSLSA 240 Wa 180 TPRATTDSSSTANLNNISITIHSEFYIIPRSQESKCNEYINNGLPPIQNTRNVVPLPLSS 239 341 342 343

The differences in amino acid compositions are colored to show the differences in amino acids at positions R101, H155, Y188, Y189, and S190, between OSU (sialidase-sensitive) and Wa (sialidase-resistance).

344 16 ACS Paragon Plus Environment

Page 17 of 32


345

PGM-MB binding assay. To determine if UVA-visible light irradiation affected VP4 protein

346

function for OSU or Wa, we developed a binding assay to quantify RV binding to cellular

347

receptors present in porcine gastric mucins coated on magnetic beads. The reason for developing

348

this method was that attempts at developing binding assays using host cells could not distinguish

349

between specific and non-specific binding of viruses because a high percentage of the RV

350

population binds non-specifically to host cells.42 In contrast, this PGM-MB binding assay uses

351

magnetic beads coated with porcine gastric mucins, which contains 0.5 to 1.5% sialic acid. These

352

mucins also contain integrins and histo-blood group antigens to bind to norovirus and Tulane

353

viruses.38 Thus, the presence of sialic acid, integrins, and histo-blood group antigens in the

354

porcine gastric mucins would allow for the quantification of binding properties of both RV

355

strains OSU and Wa. Moreover, the magnetic property of the beads facilitated the separation of

356

bound from unbound viruses as an indicator of transformation of VP8* residues important for

357

binding to host cell receptors. Virions (either with or without the irradiation treatment) were

358

incubated with mucin-coated beads, the beads were magnetically concentrated, and then

359

thoroughly rinsed before being subjected to RNA extraction. RT-qPCR was used to quantify the

360

virions with intact VP4, as an indicator of RVs that retained their ability to bind to host cell

361

receptors.

362

We conducted two types of calibration using untreated virions to ensure that this assay was

363

sensitive enough to accurately quantify changes in the number of RV bound to the PGM-MB

364

beads. First, we conducted infectivity and RT-qPCR assays for Wa and OSU without the binding

365

step to determine the reliable range for both assays. For both Wa and OSU samples, we found a

366

linear correlation between the log10 of the infectivity and log10 of the targeted RNA, as shown in

367

Figure 1a. The Pearson’s coefficients, p-values, and coefficients of determination for both the



Wa correlation and the OSU correlation were 0.98, 10-13, and 0.97, respectively. These two linear

369

correlations have similar slopes (0.75±0.03 for Wa and 0.74±0.03 for OSU), but different

370

intercepts (3.7±0.1 for Wa and 2.7±0.1 for OSU), probably due to the presence of a higher

371

number of non-matured Wa virions. The results of correlation analysis suggest that there was a

372

linear correlation between the genes detected by RT-qPCR and the PFUs present when 10 to 106

373

PFU/mL or 104 to 108 copies/mL for Wa and 100 to 107 PFU/mL or 103 to108 copies/mL for

374

OSU were present.

Log10 RNA in solutions

368

8

6

4

a 2

4

6

8

Log10 infectivity Human Wa (G1)

Porcine OSU (G5)

375


Page 18 of 32

376


Log10 RNA detected after binding

Page 19 of 32

8

6

4

b 2

4

6

8

Log10 infectivity Human Wa (G1) Porcine OSU (G5)

377

Figure 1a. Correlations between log10 infectivity in PFU/mL and log10 of targeted RNA

378

determined by RT-qPCR in copies/mL for Wa and OSU without the binding assay. Figure 1b.

379

Binding assay calibration showing the correlations between log10 of the dilution factors and log10

380

of targeted RNA determined by RT-qPCR in copies/mL. All data above the detection limits for

381

infectivity and qPCR assays were used for the correlations.

382 383

Second, we conducted a binding assay for untreated Wa and OSU viruses to determine the range

384

of virions that can be used for the binding assay. For example, if too few virions were added to

385

the beads, then PCR assay results may not be consistently reliable to quantify the bound virions.

386

Oppositely, if too many virions were present, the number of available binding sites on the beads

387

would limit virion attachment, which could lead to faulty interpretation of results. As shown in

388

Figure 1b, a linear correlation was found between the infectivity of the untreated viral samples

389

and the PCR results obtained after the binding assays for the range of 10 to 106 PFU/mL and

390

104.3 to 107 RNA copies/mL for Wa, and 102 to 107.3 PFU/mL and 104.4 to 107.9 RNA copies/mL 19 ACS Paragon Plus Environment


391

for OSU. The same correlation for both strains has the Pearson’s coefficient and the coefficient

392

of determination (R2) at 0.97 and 0.95, respectively. Based on the statistical analysis, we

393

conducted all binding assays for all virus strains within these ranges for infectivity and PCR

394

signals.

395 396

Degradation of chemical probes in sensitizer-free phosphate buffered solution (PBS) and

397

secondary effluent wastewater (SEW). To test the hypothesis that inactivation mechanisms of

398

RV due to UVA-Visible light irradiation depend on both RV genotype and the reactivity of

399

reactive oxygen species (ROS) with RV capsid, we conducted irradiation experiments using

400

either PBS or SEW. The lack of organic matter in PBS allowed the study of RV inactivation only

401

by direct effects of UVA-Visible light irradiation, while the formation of ROS from the organic

402

matter in SEW allowed the study of capsid damage due to the reaction between ROS with the

403

capsids. We first confirmed a lack of radicals present in PBS under irradiation conditions; there

404

was no significant change (p>0.05) in the concentrations of two probe compounds, phenol and

405

FFA, before and after the UVA-Visible light irradiation of PBS. In contrast, due to the presence

406

of organic matter, SEW had a quenching effect of 1.06 compared to PBS. There was a significant

407

change (P

Inactivation Mechanisms of Human and Animal Rotaviruses by Solar

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