engleski

Implementation of rat-based mumps virus neurovirulence test in quality control laboratory

Neurovirulence testing of mumps virus seeds in monkeys during the production of mumps vaccines, as it is required by cPh.Eur, can not reliably discriminate neurovirulent from non-neurovirulent mumps virus strains. A rat-based neurovirulence (RNVT) safety test was developed (JVirol 74:5382-84, 2000). An interlaboratory collaborative study initiated between FDA and NIBSC (Phase I study) demonstrated the robustness and predictability of the assay (JID 191:1123-28, 2005). This novel test avoids experiments on non-human primates taken from the wild. Rats which are to be used in tests belong to the species listed in Annex I of Council Directive 86/609/EEC. However, during implementation of the test in the quality control laboratory several difficulties were noticed. To enable comparison of RNVT results between different laboratories it is recommended that virus concentration for inoculation shall be expressed in PFU. Vaccine manufacturers mostly express mumps virus concentration in CCID50 and introduction of alternative test for virus potency would be expensive and time consuming. Moreover, we tested six mumps viruses for the ability to form plaques. Not all of tested viruses, irrespective of their genotype and attenuation status, produced readily countable plaques. Some of the mumps viruses indicated were only approximately assayable by the plaque technique because of the formation of secondary and/or nondiscrete plaques. This feature could influence the mumps virus content determination by plaque formation assay. Further, there is no unique positive control for PFUs available. In-house reference standard, calibrated to the International Reference Reagent, should be included on every plate tested and the titre of the positive control should be monitored to ensure that it maintains its level over time i.e. trend monitoring. However, regarding the differences between the ability of mumps viruses to form plaques, it is unlikely that fixed virus titres could be preassigned for common use. For above reasons we inoculated newborn rats with mumps viruses which titres represent working estimate calculated from CCID50. But, we also showed that viral inocula most likely contained more than 100 PFU/10 μl. Our preliminary results showed that brains of rats inoculated with three out of six tested mumps viruses had moderately enlarged ventricles occupying 7.3% to 16.1% of the total brain cross-sectional areas. RNVT score for wild type was lower than for some attenuated and partially attenuated viruses. It is possible that wild type virus has low neurovirulent potential as it was isolated from urine of a patient with no CNS involvement. For future validation studies, we propose that “high” virulence reference virus must be assessed on the basis of its neurovirulence potential, irrespective of its genotype. Finally, the nature of comparator in the test is obscure: “low” virulence reference virus should be recommended. Limits of detection for neurovirulence scores of attenuated strains and wild-type mumps viruses should be determined.

neurovirulence test; quality control; mumps virus

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