Microsoft word - rci inactivation of avian influenza h5n8 _2_.doc

RCI Inactivation of Avian Influenza

The influenza virus, a member of the viral family Orthomyxoviridae, is characterized as
being an enveloped single stranded negative sensed RNA virus (6) that can result in
yearly endemic outbreaks and more severe world-wide pandemic outbreaks. Influenza A
commonly infects human, swine, equine, and avian isolates. In the case of a pandemic
outbreak, highly pathogenic avian influenza (H5N1) is currently the greatest threat due to
current epidemic status in Asia, Europe, and Africa and continued threat for pandemic
spread. Reassortment of genomic information of the influenza virus can result in a more
pathogenic and infectious isolate is heightened during ongoing outbreaks, which could
result in a devastating human-to-human transmissibility. Influenza virus is typically
spread via aerosols, large droplets, or contact with infectious secretions or fomites (4).
Rapid containment of an outbreak is important for preventing further spread and
minimizing the potential for reassortment to occur. Influenza has been shown to survive
on nonporous surfaces for up to 48 hours and on material surfaces such as cloth, paper, or
tissue for up to 12 hours after being deposited at approximately a 105 TCID50/ml level
(1). In addition to surface sanitation and disinfection regimens, airborne inactivation of
influenza virus is also vital to address predominant modes of transmission such as aerosol
and large droplet (4). Environmental contamination with aerosolized droplets containing
this pathogen can serve as a reservoir for infection and must be controlled by effective
sanitation and disinfection protocols. Minimizing the degree of environmental
contamination with highly effective decontamination measures would aid in the overall
containment efforts of an outbreak.
The purpose of this study is to validate the complete inactivation of influenza A viruses
using a low pathogenic avian influenza (H5N8) as a surrogate virus for the highly
pathogenic avian influenza (H5N1) following exposure to the Radiant Catalytic
Ionization-Cell™ (RCI-Cell™) system. The RCI-Cell™ system is an advanced oxidation
tool which combines UV inactivation in the presence of hydroxical radicals so that
synergy between two highly effective inactivation technologies occurs. Efficacy will be
determined for dried inoculum on solid surfaces, in cell culture propagated inoculum, and
nebulized in a controlled chamber. Efficacy will be determined by reduced or complete
loss of infectivity in a cell culture system for treated samples compared to non-treated
positive control samples.
Virus and cells. Low pathogenic avian influenza H5N8 (H5N8, provided generously by
the Centers for Disease Control and Prevention, Atlanta, GA) was propagated in 10 day
embryonated hen eggs (Kansas State University Department of Poultry Science,
Manhattan, KS) to approximately 107 log10 TCID50 (as determined in Madin Darby
Canine Kidney, MDCK cells). Cells were maintained in Minimal Essential Medium with Earle’s salts and L-glutamine (Invitrogen Corporation, Carlsbad, CA) and 2.2 g/L sodium bicarbonate (Fisher Scientific, Hampton, NH) collectively referred to as MEM containing 10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT) supplemented with antibiotics [2.5 mg/L amphotericin B; 0.67 g/L streptomycin; and 0.3 g/L penicillin G (all from Fisher Scientific)]. Infectivity media was made by adding MEM with the addition of 0.1% TPCK treated trypsin (Fisher Scientific) and supplemented with
antibiotics (2.5 mg/L amphotericin B; 0.67 g/L streptomycin; and 0.3 g/L penicillin G).

H5N8 inactivation.
Type 302 stainless steel (McMasterCarr, Altanta, GA) coupons (2 x
10 cm2, thickness 0.8 mm) were sterilized by autoclaving for 15 min at 121 C. In a
biosafety class II cabinet, 100 μl of egg propagated H5N8 was added to each test coupon
and spread to cover the entire surface using the pipette tip and allowed to dry completely
for approximately 10-15 min. Then, the inoculated coupons were placed into a sterile
transport container and transported to the test chamber. The test coupons were then
attached to clips within the test chamber so that all sides of the coupon would be exposed
to the RCI-Cell™ treatment. One coupon was removed prior to starting the RCI-Cell™
treatment to be used as the initial control sample. The RCI-Cell™ device was then
turned on and samples were taken at various intervals (2, 4, 8, 12, 24 hours) by removing
a test coupon and preparing it for virus recovery as described below.
Virus Recovery. H5N8 virus was recovered from the stainless steel surfaces by adding
the test coupon to a sterile 50 ml conical vial (Fisher Scientific) containing 5 ml
infectivity media. Tubes were then vortexed for 1 min. Endpoint dilution titration was
conducted in MDCK cells by adding 220 µl from the 5 ml infectivity media containing
any suspended virus to the first dilution well in a minimum of 6 wells of a 96 well
microtiter plate containing confluent MDCK cells. Then, serial 1:10 dilutions were
prepared by adding 20 µl from the first well into the next 6 wells each containing 180 µl
infectivity media. The final well contained only 200 µl infectivity media to serve as a
negative cellular control. Plates were incubated at 37 C, 5% CO2 for 48 hours.
Cytopathic effect (CPE) was determined for each well and viral counts were reported as
TCID50/ml as calculated by Reed and Muench (3).
Real-Time Reverse Transcription Polymerase Chain Reaction (rRT-PCR). Viral
RNA was recovered using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA).
Quantitative detection of the extracted influenza RNA was conducted using rRT-PCR
using a fluorescently labeled TaqMan probe. The rRT-PCR primer and probe sequences
were provided generously by the Molecular Genetics Influenza Branch, Centers for
Disease Control and Prevention in Atlanta, GA. The detection threshold for successfully
detecting influenza RNA was a FAM fluorescence signal ≥ 3 using the SmartCycler.


The average amount of H5N8 recovered from the stainless steel coupons in all
experiments was 5.35 log10 TCID50/ml. Following treatment with the RCI-Cell™, the
average log reductions of the H5N8 virus were 1.85, 2.79, 4.16, 5.35, and 5.35 log10 TCID50/ml following 2, 4, 8, 12, and 24 hour treatments (Figure 1) based on the recovery Figure 1: Recovery of H5N8 post-treatment with RCI-Cell™ based on TCID50/ml in MDCK cells.
The average amount of viral H5N8 RNA recovered from the stainless steel coupons in all experiments was 4.00 log10 based on a quantitative RT-PCR available for influenza A viruses. Following treatment with the RCI-Cell™, the average log reductions of the H5N8 virus based on the amount of RNA recovered varied between 0.23 to 0.54 log10 following all exposure times (2, 4, 8, 12, and 24 hour) indicating that the mechanism of action for loss of infectivity was more likely due to disruption of the lipid envelope or structural proteins than with degradation of the viral nucleic acid (Figure 2). Figure 2: Recovery of H5N8 RNA post-treatment with RCI-Cell™ based on quantitative RT-PCR.

In an effort to better understand the inactivation of the influenza virus using the RCI-
Cell™, the efficacy was evaluated using a low pathogenic avian influenza isolate, H5N8
inoculated onto stainless steel surfaces. Inactivation efficacy was determined following
the current EPA guidelines for determining virus disinfection (2) which allows the recovery of treated virus as endpoint dilution including a TCID50 recovery assay of infectious virus. In addition to the recovery of infectious virus, we wanted to determine if any disruption of viral RNA was occurring by using a quantitative RT-PCR assay specific for influenza A viruses in our experiments. Based on the current EPA guidelines to achieve a > 4.0 log10 reduction in starting virus titer (2), RCI-Cell™ treatment for 8 hours or more resulted in the successful inactivation of the H5N8 isolate (Figure 1) for a starting contamination level of 5.35 log10 TCID50/ml. Additional testing would be required to determine if lower exposure times would result in complete inactivation for contamination levels lower than 5.35 log10 TCID50/ml, which might be more representative in a real outbreak (1, 5).
The quantitative RT-PCR results indicate that degradation of viral RNA (Figure 2) was
not the major mechanism for viral inactivation, as the levels of RNA recovered after each
treatment time were not significantly different from each other, P > 0.05. Other possible
viral targets include the lipid envelope and structural proteins which were likely affected
by the RCI-Cell™ treatment. The oxidative mechanism of this treatment likely disrupted
the relatively susceptible envelope and could have resulted in denaturing the surface
structural proteins of the influenza virus necessary for successful attachment and entry
mechanism vital for infectivity.
The results obtained in this research experiment show that exposure to the RCI-Cell™
system for 8 hours results in the required level of inactivation of an avian influenza
isolate, H5N8 which was used as a safe surrogate for the highly pathogenic H5N1 isolate.
The mechanism of action of this technology is likely due to the oxidative chemistry
resulting in both disruption of the lipid envelope and the denaturing effect on the
structural viral proteins necessary for virus replication.

Bean, B., B. M. Moore, B. Sterner, L. R. Peterson, D. N. Gerding, and H. H.
J. Balfour.
1982. Survival of Influenza Viruses on Environmental Surfaces. The
Journal of Infectious Diseases 146:47-51.
EPA 2005, posting date. Antimicrobial Science Policies Disinfectant Technical
Science Section. [Online.]
Reed, L. J., and H. Muench. 1932. A simple method for estimating 50%
endpoints. American Journal of Hygiene 27:493-497.
Tellier, R. 2006. Review of Aerosol Transmission of Influenza A Virus.
Emerging Infectious Disease 12.
WHO. 2006. Nonpharmaceutical Interventions for Pandemic Influenza,
International Measures. Emerging Infectious Disease 12:81-87.
Wright, P. F., and R. G. Webster. 2001. Orthomyxoviruses, Fourth ed, vol. 1.
Lippincott Williams & Wilkins, Philadelphia.


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