Jpn. J. Infect. Dis., 52, 1999

Laboratory and Epidemiology Communications

Difference of Polio Virus Isolation from Stool Specimens in Different Cell Lines

Akio Hagiwara*, Tetsuo Yoneyama, Kumiko Yoshii, Hiromu Yoshida, Hiroyuki Shimizu and Tatsuo Miyamura

Laboratory of Enteroviruses, Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011

The program of global eradication of poliomyelitis is ongoing. In the Western Pacific Region, there have been no wild polioviruses isolated from paralytic patients since March of 1997. The transmission of wild polioviruses seems to be effectively interrupted (1).

During the examination of stool specimens from countries where wild polioviruses still exist, we noted discrepancies among different cell lines. Table 1 shows virus isolation from stool specimens collected in Cambodia during 1996-97; three cell lines, RD, HEp-2, and Lα cells are included (2). There were some differences in the poliovirus isolation results. As for the isolation of wild polioviruses, the number of isolates was 21, 25, and 23 in HEp-2, RD, and Lα cells, respectively. There were also some differences in the number of vaccine-strain isolates among examinations of different cell lines, as shown in Table 1 (the total number of isolates was 13 in HEp-2 cells, 12 in RD cells and 14 in Lα cells). To investigate the reason for this discrepancy, we measured poliovirus titers in stool extracts. Seven stool specimens positive for type 1 wild poliovirus were selected. Of the seven specimens, three were positive for wild poliovirus in all three cell lines; however, the other four specimens were negative as shown in Table 2. Cam108, one of our specimen codes (Cam: specimen from Cambodia;108: the serial number in our laboratory), was negative for wild poliovirus in HEp-2 and Lα cells, but was positive in RD cells. Cam122 and SV1976 (SV: southern Vietnam) were positive for the poliovirus, but only in Lα cells. Cam124 was positive in HEp-2 and RD cells, but not in Lα cells. These four specimens had lower titer of poliovirus than other three samples; the titers of Cam 122, Cam124, and SV1976 were especially low (10^0.75, 10⁰ and 10⁰ CCID50/100 ul of stool extract, respectively). In contrast, in samples exceeding 10² CCID50 of poliovirus in 100 ul of stool extract, the poliovirus was easily isolated in RD, HEp-2, and Lα cells.

At the final stage of the polio eradication program, the sensitivity of detecting poliovirus in stool specimens is extremely critical. As far as poliovirus isolation is concerned, the number of polioviruses isolated in RD cells was higher than that of isolates in HEp-2 or Lα cells. However, as mentioned above, the virus titer might be related to the efficiency of virus isolation. The low titer of poliovirus in stool extracts might explain the differences among the tests. In addition, enteroviruses may interfere with the replication of polioviruses and may explain inconsistent results; this would be the case, for example, with SV1976, in which wild poliovirus was mixed with other nonpolio-enterovirus. When the titer of poliovirus in stool specimens is low, it may be necessary to use multiple cell lines for virus isolation. In addition, when poliovirus and non-polioviruses are present in stool specimens, it is recommended that Lα or L20B cells are used for selective isolation of poliovirus. L20B is another poliovirus receptor expressing the murine cell line (3, 4). Both types of cell are sensitive to polioviruses alone, and not sensitive to other enteroviruses.

REFERENCES

1. Hagiwara, A., Yoneyama, T., Yoshii, K., Yoshida, H., Shimizu, H., Wada, J., Thanh, N. T. H., Tu, P. V. and Miyamura, T. (1999): Genetic analysis of wild polioviruses towards the eradication of poliomyelitis from the Western Pacific Region. Jpn. J. Infect. Dis., 52, 146-149.
2. Koike, S., Horie, H., Ise, I., Okitsu, A., Yoshida, M., Iizuka, N., Takeuchi, K., Takegami, T. and Nomoto, A. (1990): The poliovirus receptor protein is produced both as membrane-bound and secreted forms. EMBO J., 9, 3217-3224.
3. Meldelsohn, C. L., Wimmer, E. and Racaniello, V. R. (1989): Cellular receptor for poliovirus: Molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell, 56, 855-865.
4. Pipkin, P. A., Wood, D. J., Racaniello, V. R. and Minor, P. D. (1993): Characterization of L cells expressing the human poliovirus receptor for the specific detection of poliovirus in vitro. J. Virol. Methods, 41, 333-340.
   

*Corresponding author: E-mail: ahagiwar@nih.go.jp

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