Elsevier

Biologicals

Volume 31, Issue 3, September 2003, Pages 181-185
Biologicals

Stability of minute virus of mice against temperature and sodium hydroxide

https://doi.org/10.1016/S1045-1056(03)00037-XGet rights and content

Abstract

Treatment with steam and/or dilute NaOH are commonly used techniques to disinfect manufacturing vessels and tools in the pharmaceutical industry. The aim of this procedure is sanitisation and inactivation of microbiological and viral contaminants. Here we describe the inactivation of the mouse parvovirus Minute Virus of Mice (MVM) under these conditions. Parvoviruses are known to be resistant to physico-chemical treatment and one representative of this family, the human parvovirus B19, is a potential contaminant of blood plasma. We show inactivation kinetics for MVM treated with wet-heat (70, 80, 90 °C) and with 0.01–1 M NaOH solutions (pH ⩾11.9). Robust inactivation was only achieved at 90 °C for at least 10 min and in NaOH solutions of pH ⩾12.8 (0.1 M NaOH). It was observed, that aggregation of viruses might protect viral particles from inactivation by NaOH. Therefore, appropriate sample preparation of spiking material is important for accurate simulation of the naturally occurring situation. The observed stability at pH 11.8 exceeds the previously reported upper limit of pH 9. Inactivation was due to disintegration of the viral capsid as assessed by accessibility of viral DNA for endonucleases.

Introduction

Several barriers are implemented to exclude virus contaminated donations from the plasma pool. However, due to the window period, where detectable virus or antibody is absent, a certain risk for including contaminated plasma remains. There is also the risk of adventitious viruses, which are introduced into the process by other means than the raw material. Another peril is the arising of emerging viruses, for which there are currently no tests. Therefore the plasma processing industry as well as other types of pharmaceutical industries continue to invest in the introduction of new pathogen inactivation/removal steps in their production processes. Recently, increasing attention has beenfocused on establishing efficient cleaning procedures to prevent carry-over from batch to batch. Therefore production lines are outfitted with cleaning-in place (CIP) devices. Treatment with steam and/or NaOH solution is a commonly used technique for such CIP devices. However, literature on stability of viruses under these conditions is scarce [1], [2], [3]. To improve this unsatisfactory situation we decided to test the efficiency of some of these procedures regarding virus inactivation.

We have chosen MVM as a model virus because (i) it is known to be highly resistant to physico-chemical treatment [4]and (ii) it belongs to the family of Parvoviridae (small, non-enveloped, DNA-virus), the same family to which parvovirus B19 (potential contaminant in human plasma) belongs. Textbooks [5]describe parvoviruses to be resistant to heat, detergents, organic solvents, and exposure to pH 3–9. Parvoviruses can be inactivated by formalin, beta-propiolactone,hydroxylamine, and oxidising agents [6]. Because of their robustness parvoviruses are the model of choice to validate the above mentioned inactivation treatments.

The fact that steam treatment of a surface leads to condensation and warming-up of the material, resulting in a wet and hot surface, justifies the use of the following approach to simulate the wet and hot environment encountered during steaming on the vessel surface: MVM was incubated in H2O for a determined time in a temperature range of 70–90 °C.

Another commonly used technique to disinfect vessels is rinsing with 1 M NaOH. Inactivation of MVM by NaOH was achieved by spiking the virus into NaOH solutions of various concentrations and incubation for a given period.

Virus inactivation can be due to complete disintegration of the capsid or to rather subtle structural changes in the receptor-binding motif on the capsid surface, or in destruction of the viral genome. To further investigate the inactivation mechanism we tested virion integrity by endonuclease treatment. Accessibility of viral DNA by mung bean nuclease (39 kD on SDS–PAGE [7]) was used as a measure for capsid integrity. Intact MVM–DNA was quantified by real-time PCR.

We showed that the parvovirus MVM is stable up to at least pH 11.9 and up to temperatures of 80°C for one hour in a wet environment. Treatment at pH 12.8 and above, or 90 °C for at least 1 min or 10 min, respectively resulted in >4 log inactivation. The mechanism of inactivation is due to disintegration of the viral capsid.

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Cell culture dishes and reagents were purchased from Becton Dickinson, Franklin Lakes, NJ, USA and Sigma, Buchs, Switzerland; oligos were synthesised at Microsynth (Balgach, Switzerland).

MVM cultivation

A9 cells (ECACC: 85011426) were infected at 10% confluence with MVM (p-strain; ATCC: VR-1346). After total lysis of the cell layer, virus was harvested from the supernatant and from cell debris. Cell debris was exposed to three freeze–thaw cycles to release trapped virus.

Cell culture

The A9 mouse fibroblast cell line was

Temperature

To test the resistance of MVM towards wet-heat as encountered by steaming on vessel surfaces we spiked virus into H2Oddequilibrated at the corresponding temperatures (70, 80, 90 °C). Samples were withdrawn after 0, 1, 2, 5, 10, 20, and 60 min, and immediately cooled on ice until titration.

Fig. 1shows the inactivation kinetics of MVM at the indicated temperatures. MVM is stable at 70 °C for one hour, even at 80 °C only a moderate decrease in infectivity is observed. A robust inactivation (⩾4.7

Discussion

The relative stability of parvoviruses is due to the lack of a lipid envelope, their small size, and simple construction. Parvoviruses encapsidate their ssDNA genome with 60 copies of a combination of VP1, VP2, and VP3 proteins. Fifty-four copies are VP2 or VP3. VP3 differs from VP2 only by the cleavage of 19 amino acids from the VP2 N-terminus [15]. VP1 and VP2 are formed by alternative splicing. VP1 shares its C-terminus with VP2 but carries a 143 amino acid long sequence at its N-terminus

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