Vaccine 19 (2001) 1456 – 1459 Analysis of the accumulation of mutants in Sabin attenuated polio vaccine viruses passaged in Vero cells Hitoshi Horie a, *, Miwako Miyazawa a , Yoshihiro Ota a , Kengo Wakabayashi a , Hiromu Yoshida b , Yutaka Doi a , So Hashizume a a Japan Poliomyelitis Research Institute, Kumegawa -cho 5 -34 -4, Higashimurayama, Tokyo 189 -0003, Japan b Department of Virology II, National Institute of Infectious Diseases, Gakuen 4 -7 -1, Musashimurayama, Tokyo 208 -0011, Japan Received 15 May 2000; received in revised form 17 August 2000; accepted 8 September 2000 Abstract To confirm the safety of oral poliomyelitis vaccine (OPV) cultured in Vero cells, the genetic stability of cultured polio vaccine viruses was analysed by MAPREC (mutant analysis by PCR and restriction enzyme cleavage). The rates of mutant accumulation of the viruses passaged in Vero cells under a low multiplicity of infection (MOI) condition (approximately 10 -3.5 CCID 50 /cell; the same as under usual OPV production conditions) were higher than those passaged in secondary cultured monkey kidney cells. However, the rates of mutant accumulation were restrained when the viruses were cultured under a high MOI condition (approximately 10 -1.5 CCID 50 /cell) in Vero cells. Furthermore, neurovirulence of the passaged viruses in pollovirus susceptible transgenic mice PVR-Tg21 was shown to correlate highly with the results of MAPREC. It is expected that our results will contribute to the large scale preparation of safe and effective OPV using Vero cells. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: OPV; Vero cell; MAPREC www.elsevier.com/locate/vaccine Oral poliomyelitis vaccine (OPV) is considered to be the major tool for worldwide eradication of poliomyeli- tis [1–3]. OPV has been produced by culturing Sabin vaccine seed viruses in kidney cells from rhesus, green, and patas monkeys in the world [4]. However, the use of the monkeys has many disadvantages: a high risk of zoonosis due to the close evolutionary relatedness to humans, the problem of species extinction in nature, extremely high breeding costs, and marked individual variations in vaccine production. Therefore, a new cell substrate to replace the monkey kidney cells is neces- sary. The Vero cell line, which is one of the continuous cell lines and is highly useful internationally, is expected for OPV production. However, we have experience that Sabin viruses cultured in Vero cells contain more viru- lent mutants than those cultured in primary monkey kidney cells (unpublished data). In the present study, to confirm the safety of OPV cultured in Vero cells as well as in primary monkey kidney cells, we analysed the genetic stability of cul- tured Sabin vaccine viruses in Vero cells [the origin of our Vero cells was the ATCC (American Type Culture Collection)] and secondary green monkey kidney (sGMK) cells with the MAPREC (mutant analysis by PCR and restriction enzyme cleavage) method. The method was originally designed by Chumakov et al. [5–7] and modified by Horie et al. [8] to estimate the ratio of viruses containing genes of a virulent nature in a vaccine virus population. Specific bases which are involved in neurovirulence on Sabin virus gene were analysed: in the case of type 1 with a virulent base A at position 480 and base C at position 525, for type 2 with a virulent base G at position 481, for type 3 with a virulent base C at position 472. The Sabin vaccine viruses passaged in Vero or sGMK cells with a virus concentration of approximately 10 -3.5 CCID 50 /cell and incubated at a temperature of 33.3°C (the same as under usual OPV production conditions) were analysed by MAPREC. Percentages of 480-A +525-C and 472- C, which were obtained from type 1 and type 3 Sabin vaccine viruses passaged three to five times in sGMK * Corresponding author. Tel.: +81-423-93-3191; fax: +81-423-92- 7885. 0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII:S0264-410X(00)00350-9