Expert report on virucidal activity of Bio 5e against bovine viral diarrhea virus

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1 Dr. Jochen Steinmann Mikrolab GmbH D Bremen Norderoog Dr. St/sbe Expert report on virucidal activity of Bio 5e against bovine viral diarrhea virus (surrogate of HCV) Examinations of virucidal activity of chemical disinfectants against hepatitis C virus (HCV) are not possible, since there are no in vitro tests available. Therefore, bovine viral diarrhea virus (BVDV) serves as a model virus, because this pestivirus also belongs to the family Flaviviridae and has some properties similar to HCV (1,2,3). BVDV is a small ssrna virus with a nonsegmented positive strand and has before often used in blood product industry as a surrogate for HCV. This study was carried out in accordance with the guideline on testing chemical disinfectants for effectiveness against viruses published by the Federal Office of Health (Bundesgesundheitsamt, BGA, now Robert Koch-lnstitut, Berlin, Germany) and the German Association for the Control of Viruses Diseases (Deutsche Vereinigung zur Bekampfung der Viruskrankheiten, DW) (4,5).

2 1. Identification of test laboratory Mikrolab GmbH, Norderoog 2, D Bremen 2. ldentification of sample Name of the product Bio 5e Lot no. ESS3A Appearance and smell of the product clear, yellowish fluid; product specific Expiry time - ph-values undiluted: 9.21 (20 C) Date of receipt at laboratory 2003-I O-21 Conditions of storage room temperature in the dark Active substances and quats, alkylamines concentrations 3. Experimental conditions period of analysis test temperature 20 C ± 1 C product test concentration 0.50 % Contact times 5, 10 and 15 minutes interfering substance bovine serum albumin (BSA) fetal calf serum (FCS)

3 diluent of product test virus Aqua bidest. BVDV strain NADL 4. Material and methods 4.1 Preparation of virus suspension BVDV strain NADL (VR-534) originated from Dr. Frey, Institute of Virology at the School of Veterinary Medicine Hannover (Tiertirztliche Hochschule, D Hannover). Before inactivation assays, virus had been passaged 5 times in KOP-R (primary cells from bovine oropharyngeal tissue) and once in primary bovine kidney cells. KOP-R cells originated from the Bundesforschungsanstalt fur Viruskrankheiten der Tiere, isle of Riems (Dr. R. Riebe, catalog No. RIE 244).

4 For preparation of virus suspension, KOP-R cells, which were cultivated with Eagle s Minimum Essential Medium (EMEM; Cambrex Bio Science Verviers s.p.r.l., B-4800 Verviers, Belgium) supplemented with I-glutamine, sodium pyruvate and 10% or 2% fetal calf serum (FCS, Biochrom AG, D Berlin, Germany; antibodies against BVDV not detectable), were infected with the BVDV. After cells showed a constant cytopathic effect, they were subjected to a rapid three-fold freeze-thaw procedure. This was followed by lowspeed centrifugation (10 min and 1000 x g) in order to sediment cell detritus. After aliquotation, virus suspension was stored at -80 C. 4.2 Inactivation tests The tests were carried out in accordance to the BGA and DW guideline. Eight parts by volume of the disinfectant (1.25 x desired concentration) were mixed with one part by volume of virus suspension and one part by volume of double distilled water. In tests with interfering substances, instead of double distilled water, one part by volume of bovine serum albumin (BSA, Sigma-Aldrich Chemie GmbH, D Taufkirchen) or one part by volume of fetal calf serum was added. Inactivation tests were carried out in sealed glass test-tubes in a water bath at 20 C ± 1 C. Aliquots were removed after the appropriate exposure times, and residual infectivity was determined. To reduce cytotoxicity, immediately at the end of the exposure time the mixture was added to a MicroSpin S 400 HR column (see 7. elimination of cytotoxicity) and centrifuged. In addition, in accordance with the guideline, virus controls were carried out. 4.3 Determination of infectivity Infectivity was determined by means of end point dilution titration in a micro-procedure. For

5 this, samples were diluted with ice-cold EMEM and 100 µl of each dilution were placed in 8 wells of a sterile polystyrene flat bottomed plate (Nunc A/S, DK-4000 Roskilde, Denmark). 100 µl of a fresh trypsinated KOP-R cell culture (passage 28 th - 35 th ) were added. The suspension was adjusted to reach approximately x 10 3 cells per well. Incubation was at 37 C in a CO 2 -atmosphere (5.0% CO 2 - content). Finally, cultures were observed for cytopathic effects for five days of inoculation. The infectious dose (ID 50 ) was calculated according to the method of Spearman (6) and Karber (7).

6 4.4 Elimination of cytotoxicity Since the cytotoxicity did not allow to follow the reduction of residual infectivity titre over the range of four log 10 ID 50, ready to use MicroSpin S 400 HR columns (Amersham Biosciences Europe GmbH, D Freiburg, Germany) were used according to the instructions of the manufacturer with addition of a 0.3 % BSA solution before loading. 4.5 Determination of cytotoxicity For determination of cytotoxicity of the disinfectant, two parts by volume of PBS were mixed with eight parts by volume of the disinfectant, diluted with ice-cold EMEM and inoculated into cell culture. These tests were also performed with MicroSpin columns. 4.6 Calculation of the virucidal effect The virucidal effect of the test disinfectant was evaluated by calculating the decrease in titre in comparison with the control titration without disinfectant. The difference is given as reduction factor (RF) or A log ID Results In parallel with the inactivation tests, cytotoxicity of Bio 5e was measured. Examinations showed that without treatment the disinfectant had a log 10 CD 50 /ml of 3.5, whereas no cytotoxic effect after treatment with the columns was measured resulting in a log 10 CD 50 /ml of 1.5 (data not shown in table). These tests to measure cytotoxicity are imperative, because in this way the lower detection threshold for non-inactivated BVDV is determined.

7 Results of inactivation tests are found in table 1. Exposure times were 5, 10 and 15 minutes. Testing Bio 5e as a 0.50 % solution without soil load, an efficacy was measured after an exposure time of five minutes (Table 1). At that time, no BVDV was detectable any longer. The RF s were 5.12 (assay without soil load), (assay with BSA) and 4.37 (assay with FCS), respectively. This corresponds to an inactivation of % or 99.99%.

8 Summarizing the results, it can be recommended to use Bio 5e for inactivation of BVDV (surrogate of hepatitis C virus) as follows: 0.60 % 5 min

9 Literature 1. Sattar, S.A., J. Tetro, V.S. Springthorpe and A. Giulivi: Preventing the spread of hepatitis B and C viruses: Where are germicides relevant? Am J Infect Control 2001; 29: Zitzmann, N., A.S. Mehta, S. Carrouee, T.D. Butters, F.M. Platt et al.: lmino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: Implications for the development of broad spectrum anti-hepatitis virus agents. Proc Natl Acad Sci USA, 1999; 96: Jiang X, Wang M, Wang K, Estes MK. Sequence and genomic organization of Norwalk virus. Virology 1993; 195: Richtlinie des Bundesgesundheitsamtes und der Deutschen Vereinigung zur Bekampfung der Viruskrankheiten e.v. zur Prtifung von chemischen Desinfektionsmitteln auf Wirksamkeit gegen Viren. Bundesgesundheitsblatt 1982; 25: Kommentar zur Richtlinie des Bundesgesundheitsamtes und der Deutschen Vereinigung zur Bekampfung der Viruskrankheiten e.v. zur Prufung von chemischen Desinfektionsmitteln auf Wirksamkeit gegen Viren. Bundesgesundheitsblatt 1982; 25: Spearman, C.: The method of right or wrong cases (constant stimuli) without Gauss s formulae. Brit J Psycho1 1908; 2: Karber, G.: Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche.

10 Arch Exp Path Pharmak 1931; 162:

11 DR. JOCHEN STEINMANN Wiss. techn. Leiter der Mikrolab GmbH D Bremen Norderooa 2, Dr. St/sbe Wirksamkeit von Bio 5e gegenijber dem Bovine Viral Diarrhea Virus (BVDV) im quantitativen Suspensionsversuch bei 20 C GUTACHTERLICHE BEURTEILUNG Das Flachendesinfektionsmittel Bio 5e der Goldschmidt AG wurde gema Auftrag auf seine viruziden Eigenschaften gegentiber dem Bovine Viral Diarrhea Virus (BVDV) in enger Anlehnung an die Richtlinie des Bundesgesundheitsamtes (BGA, jetzt Robert Koch-lnstitut) und der Deutschen Vereinigung zur Bekampfung der Viruskrankheiten e.v. (DVV) untersucht. Das BVDV diente dabei als Surrogatvirus fur das Hepatitis C Virus (HCV), da dieses nicht im Tiermodell und in einem Zellkultursystem zur Vermehrung gebracht werden kann. Durch die Testung dieses Surrogatvirus wird die Moglichkeit geschaffen, Aussagen zu Anwendungsempfehlungen fur das zu prufende Praparat gegenuber dem HCV zu machen. In der Richtlinie des BGA und der DVV wird dann von einer Virus-Wirksamkeit eines Desinfektionsmittels ausgegangen, wenn nach einer bestimmten Einwirkzeit eine Reduktion des initialen Virustiters urn vier log,,-stufen (Inaktivierung 99,99%) et-folgt ist. Das Flachendesinfektionsmittel Bio 5e wurde als 0,50%ige Losung untersucht. Die Einwirkzeiten betrugen 5, 10 und 15 Minuten.

12 Zusammenfassend kann nach diesen Untersuchungsergebnissen empfohlen werden, das Flachendesinfektionsmittel Bio 5e zur BVDV-lnaktivierung wie folgt einzusetzen: 0,50% 5 Minuten

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14 Table 1: BVDV-Inactivating properties Bio 5e in a quantitative suspension test at 20 C Titre reduction (RF) Concentration of disinfectant Virus content in the control* (log 10 ID 50 /ml) Interfering substances after 5 min 10 min 15 min 0.50% 6.62 Without 0.50% % BSA 0.50% % FCS * determination of virus titre was performed after the longest exposure time