Inactivation of Six Strains of Turkey Reovirus by a Commercial Device Using Ultraviolet Light and Ozone

Nader M. Sobhy; Jason S. Hatfield; Sunil K. Mor; Sagar M. Goyal

doi:10.20944/preprints202510.2238.v1

Submitted:

28 October 2025

Posted:

29 October 2025

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Abstract

Turkey reoviruses (TRV) are associated with diverse clinical syndromes in turkeys. Based on the site of virus isolation, the TRVs are designated as TARV (turkey arthritis reovirus), THRV (turkey hepatitis reovirus), and TERV (turkey enteritis reovirus). The environmental stability of TRVs facilitates transmission through contaminated fomites, therefore interventions that reduce or eliminate fomite contamination are essential for biosecurity. We studied the inactivation of six TRV strains using the UVZone Shoe Sanitizing Station (UV-SSS), a commercially available device based on ultraviolet (UV) light and ozone. Viral suspensions of different TRV strains were applied to six types of fomites: stainless steel, aluminum, polypropylene, cardboard, rubber, and fabric. These fomites were exposed to either one 8-second cycle or two consecutive 8-second cycles, and the surviving virus was titrated in QT-35 cells. Inactivation varied significantly with both fomite type and exposure duration. A 16-second treatment consistently achieved greater reductions than a single cycle, with inactivation rates exceeding 90% on porous surfaces, including stainless steel, aluminum, and polypropylene. In contrast, porous materials such as cardboard and fabric showed lower reductions. Strain-specific differences were also observed, with enteric isolates exhibiting higher resistance compared to a brain-derived strain. These results demonstrate that UV-ozone disinfection can substantially reduce TRV on farm-relevant fomites, though effectiveness is influenced by material type, viral strain, and exposure time.

Keywords:

Turkey reovirus

;

shoe sanitizer

;

strains

;

fomites

;

ozone

;

ultraviolet light

Subject:

Biology and Life Sciences - Virology

1. Introduction

Implementing biosecurity practices to combat viral and bacterial infections on turkey farms is crucial to the farm's economic viability. Avian reoviruses are important pathogens of turkeys, causing tenosynovitis, arthritis, hepatitis, myocarditis, and encephalitis [1,2,3,4]. The last decade has seen a surge in turkey reovirus (TRV) cases, accompanied by the continuous evolution of the virus, which has resulted in the emergence of pathogenic variants. Currently, seven genotypic clusters of avian reoviruses have been recognized [5]. The turkey reoviruses belong to one of those clusters as defined by sigma C sequences. Based on M2 gene genotyping, however, three different genotypes of TRV have been recognized [6]. Currently, there is no commercial vaccine available to combat TRV.

Technologies to minimize viral entry onto farms are always in demand because viruses, after being shed by their infected hosts, can contaminate and survive on inanimate fomites [7]. Once contaminated, fomites serve as vehicles for the transmission of pathogens to susceptible hosts. The survival of viruses on fomites varies depending on intrinsic factors such as virus strain, initial viral load, and fomite properties, as well as extrinsic factors including relative humidity, sunlight, and temperature. These factors may enable the virus to remain viable long enough to contact new hosts. Both porous and non-porous fomites are found on poultry farms, and viruses generally survive longer on porous surfaces compared to non-porous ones, though some exceptions do exist [8].

Recently, new devices have become available that are purported to inactivate viruses in the environment. One such device is the UVZone Shoe Sanitizing Station (UV-SSS) developed by PathO3Gen Solutions, St. Petersburg, FL. This device generates ozone using ultraviolet (UV) light. Based on wavelength range (100–400 nm), UV comprises four different regions: UVA, UVB, UVC, and vacuum UV. UVA (315–400 nm) has the longest wavelength with the lowest energy, while UVB (280–315 nm) represents the medium wavelength range. UVB is often used for tanning, but has the potential to cause skin burning and even cancer. The UVC (200–280 nm) exhibits germicidal effects, while vacuum UV (100–200 nm) is transmitted only in a vacuum [9]. The UVC can inactivate fungi, viruses, and bacteria [10,11,12] by damaging the DNA/RNA of microbial pathogens and producing photo-dimers between the pyrimidine nucleotide molecules of the genome [13]. In addition, UVC lamps at a wavelength of 185 nm can generate ozone in ambient air. The generated ozone, along with UVC, produces synergistic germicidal effects against fungal, bacterial, and viral pathogens [14,15,16]. In this study, we compared the inactivation of six strains of turkey reovirus on fomites using the UVZone Shoe Sanitizing Station.

2. Materials and Methods

Viruses. Six strains of TRV, namely turkey enteritis reovirus (TERV), highly pathogenic turkey arthritis reovirus-1 (TARV-1), mutated turkey arthritis reovirus-2 (TARV-2), turkey hepatitis reovirus (THRV), a brain isolate of turkey reovirus (TBRV), and a highly pathogenic strain of TARV (TARV-3 or O’Neil strain), were used in this study. These designations (TERV, TARV, THRV, TBRV) are widely used in the field to differentiate turkey-origin reovirus isolates based on clinical presentations or tissue tropism; however, they do not represent formal taxa recognized by the International Committee on Taxonomy of Viruses (ICTV). All isolates are classified within the species Avian orthoreovirus, family Reoviridae, genus Orthoreovirus. All viruses were grown and titrated in Japanese quail fibrosarcoma (QT-35) cells. The cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 8% fetal bovine serum, 1 µg/ml fungizone, 50 µg /ml neomycin, 150 IU/ml penicillin, and 150 µg g/ml streptomycin.

Source of UV and ozone. A commercial device called the “PathO3Gen Solutions UVZone Shoe Sanitizing Station (UV-SSS)” was obtained from the manufacturer. The device has two ozone-generating lampsFigure and four UV generating lamps, measuring approximately 357 mm and 386 mm in length, respectively. The ozone generating lamps emit at 185 nm, a wavelength that photolyzes molecular oxygen (O₂) to produce ozone (O₃), while the UV-C lamps emit at 254 nm, a germicidal wavelength that directly damages nucleic acids. It is certified to be safe by a Nationally Recognized Test Laboratory. During operation, the UVZone device produces an average irradiance of approximately 2000 µW/cm² of UV-C at a wavelength of 254 nm and an ozone concentration of approximately 6.0 ppm. There are two glass shields (left and right) on the device, which are activated automatically for an 8-second disinfection cycle when someone stands on them (Figure 1). A beeping sound indicates the end of the 8-second cycle.

Fomite Materials and Preparation. Six different fomite types were tested: stainless steel (SS), cardboard (CB), rubber boot (RB), polypropylene (PP), denim fabric (FB), and aluminum (AL). Circular coupons approximately 1 cm² in area were cut using sterile scissors and placed into three separate 24-well tissue culture plates designated as the Control Plate, the Single-Cycle Plate, and the Double-Cycle Plate. The Control Plate contained fomites that were not exposed to UV or ozone, the Single-Cycle Plate contained fomites exposed to one 8-second disinfection cycle, and the Double-Cycle Plate contained fomites exposed to two consecutive 8-second disinfection cycles (total of 16 seconds exposure). Each fomite type was tested in triplicate for each plate and the entire experiment was independently repeated three times. Each coupon was inoculated with 60 µl of virus suspension and the inoculum was evenly spread across the surface using a micropipette tip. Plates were placed inside a biosafety cabinet at room temperature and left undisturbed to allow the inoculum to air dry.

Figure 1. Glass shield of PathO3Gen Solutions UVZone Shoe Sanitizing Station (SSS).

Procedure. The UVzone device was operated in a room at an ambient temperature of approximately 25ºC and 50% relative humidity. The plates with dried, inoculated fomites were placed upside down directly on the glass shield of the device, with the inoculated surface facing the UV-ozone lamps, ensuring direct exposure of the fomites to both the UV and ozone emissions. Fomites in the Control Plate were handled identically to the exposed samples and placed on the glass surface for 8 seconds without activation of the lamps to account for handling and desiccation effects. Fomites in the Single-Cycle Plate were exposed to activated disinfection cycle by placing the plate on one glass shield while stepping on the other to trigger the device. Fomites in the Double-Cycle Plate were exposed to two consecutive 8-second cycles using the same procedure. Following exposure, fomites were allowed to aerate for 2 minutes at ambient conditions to dissipate residual ozone before elution. This step was a non-chemical way to prevent potential care over effects during virus recovery.

Viruses were eluted from each fomite using 100 µl of elution buffer composed of 3% beef extract in 0.05 M glycine. The eluates were serially diluted 10-fold and were prepared in DMEM containing 2% fetal bovine serum, 50 µg/ml neomycin, 1 µg/ml fungizone, 150 IU/ml penicillin, and 150 µg/ml streptomycin. Each dilution was inoculated in triplicate onto monolayers of QT-35 cells grown in 96-well microtiter plates. The plates were incubated in a 5% CO₂ incubator at 37ºC and examined daily for 5-7 days for the appearance of virus-induced cytopathic effects (CPE). Virus titers were calculated using the Karber method [17].

UV-Ozone Source Controls. The UVZone device produces UV-C and ozone simultaneously, meaning the two disinfection mechanisms could not be independently isolated. The results represent the combined UV-ozone disinfection effect of the device under normal operating conditions.

Statistical analysis. The effect of exposure time and fomite type on the inactivation of reovirus strains was tested by two-way ANOVA. Post hoc analysis was performed for multiple pairwise comparisons for observed means using the Tukey HSD model. Significance was considered at P<0.05.

3. Results

The amount of virus inactivation was higher at 16-sec exposure than at 8-sec exposure for all reovirus strains on all fomites, although some variation was observed. As shown in Table 1, the highest inactivation of TERV was observed on AL (71.82%) and SS (63.69%) at 8 sec, and on AL (93.98%), SS (90.00 %), and PP (90.00%) at 16 sec. The lowest inactivation of TERV was observed on FB (22.37%) and CB (39.96%) at 8 sec, and CB (70.26%) and RB (78.12%) at 16 sec. At 16-sec exposure, the TERV inactivation was between 70% and 94% on various fomites (Table 1).

The highest TARV-1 inactivation was observed on AL (95.32%), SS (93.97%), and PP (83.40%) at 8 sec and on AL (98.71%), PP (97.25%), and SS (97.18%) at 16 sec (Table 2). The lowest inactivation was observed on CB (39.74%) and FB (39.74%) at 8 sec, and CB (53.23%) and FB (63.70%) at 16 sec. At 16-sec exposure, the TARV-1 inactivation was between 53% and 98.7% on various fomites (Table 2).