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Evaluation of a Decontamination Technology for Norovirus

Konstance K. Knox, PhD
Donald R. Carrigan, PhD

April 23, 2007

Wisconsin Viral Research Group
10437 Innovation Drive
Milwaukee, Wisconsin


Norwalk-like viruses (NLV) (genus norovirus) are the major cause of non-bacterial outbreaks of gastrointestinal disease in the United States and Europe with the most frequent occurrences being seen in hotels (1) and aboard cruise ships (2,3). Clinical manifestations of NLV infections include a sudden onset of nausea with projectile vomiting and watery diarrhea with the illness resolving within 72 hours. Usually the infection is self-limited, but elderly people, young children and individuals with impaired immune responses are subject to potentially severe complications due to dehydration and electrolyte disturbances.

NLV infections are most commonly transmitted by contaminated food and water (2,3) but the recurrent outbreaks that have been documented on cruise ships strongly suggest that transmission can also be associated with environmental contamination (3). In sites of a previous NLV outbreak, NLV RNA can be detected in carpets, bathroom fixtures, walls and cabinetry (1). Also, studies have demonstrated that infectious NLV can persist on fomites such as telephone handsets, computer mouse and keyboards, and metal surfaces for at least 7 days (4,5,6), and transmission from contaminated surfaces to food has been experimentally documented (5).

As could be expected given the potential public health and economic ramifications of norovirus associated disease outbreaks, numerous methods of viral decontamination have been described. These include to treatment of environmental surfaces with various kinds of chemicals such as quaternary ammonium (7), sodium bicarbonate (8), various types of alcohols (9,10,11), different formulations of chlorine or sodium hypochlorite (12,13,14), ozone (15) and both ionizing and non-ionizing radiation (16,17). In general, all of the methods described some varying degrees of efficacy, but most suffer from the impracticality of their general application to large areas such as hotel or cruise ship staterooms.

In the studies proposed here, we will use tissue culture based procedures to assess a new decontamination technology for its ability to inactivate noroviruses. This technology is easily applicable to large areas containing a variety of potentially contaminated surfaces.

The human noroviruses, including the Norwalk agent, are unable to be grown in tissue culture (18). Therefore, we will use a well established, veterinary surrogate virus for the human noroviruses, namely feline calicivirus (FCV) (19). Virtually all of the studies involving the inactivation of noroviruses cited above utilized FCV (6-1), the use of surrogate viruses in the study of virucidal compounds is well accepted (20,21) and the use of FCV as a surrogate for the human noroviruses is endorsed by the United States Environmental Protection Agency (EPA) (22).

Outline of Proposed Studies

The proposed timeline for the project can be summarized as follows:

  1. As recommended by the EPA, the Crandel Reese Feline Kidney (CRFK) line of epithelial cells will be obtained from the American Type Culture Collection (ATCC) [Manassas, Virginia] (ATCC VR-782) and established in our laboratory.
  2. Once the CRFK cells are well established, the F9 strain of FCV (also recommended by the EPA) will be obtained from the ATCC (ATCC CCL-94) and used to prepare a high titered virus stock. This stock will be aliquoted and frozen at -70oC.
  3. The virus stock be titrated using the CRFK cells by a standard plaque forming unit (PFU) assay. A detailed PFU assay using CRFK cells and the F9 strain of FCV has been published (23). In that report, which utilized materials and procedures identical to the ones that we will use, titers of FCV in several different stocks ranged from 1. 6 X 108 to 4. 4 X 108 PFU per milliliter (ml) of culture medium.
  4. For assessment of the virucidal potential of the technology, the EPA guidelines will be followed (22). In brief, 0.2 ml of virus stock will be added to replicate 100 X 20 mm sterile Petri dishes. The liquid will then be spread evenly across the dish surface with a cell scraper and allowed to dry, for 30 to 60 minutes forming a "virus film".
  5. Replicate "virus films" will then be exposed to the viral inactivation technology for various lengths of time and at various intensities. Untreated "virus films" will serve as controls.
  6. Following exposure to the virucidal treatment, two mil of tissue culture medium will be added to each dish and incubated at 37oC for one hour. The concentration of infectious virus in each sample will then be evaluated by means of the PFU assay.
  7. The efficacy of the viral inactivation procedure will be evaluated by quantitatively comparing the treated materials to the untreated controls.


  1. Cheesbrough JS, Green J, Gallimore CI, Wright PA and Brown DWG. 2000. Widespread environmental contamination with Norwalk-like viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis. Epidemiol Infect 125:93-98.
  2. Widdowson MA, Sulka A, Bulens SN, et al. 2005. Norovirus and foodborne disease, United States, 1991-2000. Emerg Infect Dis 11:95-102.
  3. Cramer EH, Forney D, Dannenberg, AL et al. 2002. Outbreaks of gastroenteritis associated with noroviruses on cruise ships - United States, 2002. MMWR 51:1112-1115.
  4. Clay S, Maherchandani S, Malik YS and Goyal SM. 2006. Survival on uncommon fomites of feline calicivirus, a surrogate of noroviruses. Amer J Infect Control 34:41-43.
  5. D'Souza DH, Sair A, Williams K, Papafragkou E, Jean J, Moore C and Jaykus L. 2006. Persistence of caliciviruses on environmental surfaces and their transfer to food. Int J Food Microbiol 108:84-91.
  6. Mattison K, Karthikeyan K, Abebe M, Malik N, Sattar SA, Farber JM and Bidawid S. 2007. Survival of calicivirus in foods and on surfaces: experiments with feline calicivirus as a surrogate for norovirus. J Food Prot. 70:500-503.
  7. Jimenez L and Chiang M. 2006. Virucidal activity of quaternary ammonium compound disinfectant against feline calicivirus: a surrogate for norovirus. Amer J Infect Control. 34:269-273.
  8. Malik YS and Goyal SM. 2006. Virucidal efficacy of sodium bicarbonate on a food contact surface against feline calicivirus, a norovirus surrogate. Inter J Food Microbiol 109:160-163.
  9. Malik YS, Maherchandani S and Goyal SM. 2006. Comparative efficacy of ethanol and isopropanol against feline calicivirus, a norovirus surrogate. Amer J Infect Control. 34:31-35.
  10. Kampf G, Grotheer D and Steinmann J. 2005. Efficacy of three ethanol based hand rubs against feline calicivirus, a surrogate for norovirus. J Hosp Infect 60:144-149.
  11. Gehrke C, Steinmann J and Goroney-Bermes P. 2004. Inactivation of feline calicivirus, a surrogate of norovirus (formerly Norwalk-like viruses) by different types of alcohol in vitro and in vivo. J Hosp Infect 56:49-55.
  12. Thurston-Enriquez JA, Haas CN, Jacangelo J and Gerba CP. 2005. Inactivation of enteric adenovirus and feline calicivirus by chlorine dioxide. Appl Environ Microbiol 71:3100-3105.
  13. Tree JA, Adams MR and Lees DN. 2005. Disinfection of feline calicivirus (a surrogate for norovirus) in wastewater. J Appl Microbiol 98:155-162.
  14. Thurston-Enriquez JA, Haas CN, Jacangelo J and Gerba CP. 2003. Chlorine inactivation of adenovirus type 40 and feline calicivirus. Appl Environ Microbiol 69:3979-3985.
  15. Thurston-Enriquez JA, Haas CN, Jacangelo J and Gerba CP. 2005. Inactivation of enteric adenovirus and feline calicivirus by ozone. Water Res 39:3650-3656.
  16. De Roda Husman AM, Bijkerk P, Lodder W, Van Den Berg H, Pribil W, Cabaj A, Gehringer P, Sommer R and Duizer E. 2004. Calicivirus inactivation by nonionizing (253. 7 nanometer wavelength [UV] and ionizing (gamma) radiation. Appl Environ Microbiol 70:5089-5093.
  17. Thurston-Enriquez JA, Haas CN, Jacangelo J, Riley K and Gerba CP. 2003. Inactivation of feline calicivirus and adenovirus type 40 by UV radiation. Appl Environ Microbiol 69:577-582.
  18. Green KY, Chanock RM, and Kapikian AZ. Huamn Caliciviruses. In Fields Virology, Fourth Edition. DM Knipe and PM Howley (eds). Lippincott, Williams and Wilkins. Philadelphia, Pennsylvania. 2001. pp. 841-874.
  19. Radford AD, Coyne KP, Dawson S, Porter CJ. and Gaskell RM. 2007. Feline calicivirus. Vet Res 38:319-335.
  20. Radford AD, Gaskell RM and Hart CA. 2004. Human norovirus infection and the lessons from animal caliciviruses. Curr Opin Infect Dis. 17:471-478.
  21. Steinmann J. 2004. Surrogate viruses for testing virucidal efficacy of chemical disinfectants. J Hosp Infect 56 Suppl 2:S49-S54.
  22. URL: http://www.epa.gov/oppad001/pdf_files/initial_virucidal_test.pdf
    URL: http://www.epa.gov/oppad001/pdf_files/confirmatory_virucidal_test.pdf
  23. Bidawid S, Malik N, Adegbunrin O, Sattar SA and Farber JM. 2003. A feline kidney cell line based plaque assay for feline calicivirus, a surrogate for Norwalk virus. J Virol Meth 107:163-167.
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