Yutaka Doi

Neurovirulence of Type 1 Polioviruses Isolated from Sewage in Japan

ABSTRACT Sixteen type 1 poliovirus strains were isolated from a sewage disposal plant located downstream of the Oyabe River in Japan between October 1993 and September 1995. The isolates were intratypically differentiated as vaccine-derived strains. Neutralizing antigenicity analysis with monoclonal antibodies and estimation of neurovirulence by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) were performed for 13 type 1 strains of these isolates. The isolates were classified into three groups. Group I (five strains) had a variant type of antigenicity and neurovirulent phenotype. Group II (four strains) had the vaccine type of antigenicity and neurovirulent phenotype. Group III (four strains) had the vaccine type of antigenicity and an attenuated phenotype. Furthermore, it was demonstrated that the virulent isolates were neutralized by human sera obtained after oral poliomyelitis vaccine (OPV) administration, and the sera of rats immunized with inactivated poliovirus vaccine. Although vaccination was effective against virulent polioviruses, virulent viruses will continue to exist in the environment as long as OPV is in use.

Analysis of the accumulation of mutants in Sabin attenuated polio vaccine viruses passaged in Vero cells

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)CCID50/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)CCID50/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.

Evaluation of an enzyme-linked immunosorbent assay based on binding inhibition for type-specific quantification of poliovirus neutralization-relevant antibodies.

To detect neutralization‐relevant antibodies against 3 types of poliovirus (PV) without using tissue cultures and live viruses, an enzyme‐linked immunosorbent assay (ELISA) based on monoclonal antibody‐binding inhibition was evaluated using sera from 80 vaccinated Japanese children and 60 Pakistani poliomyelitis patients. Compared with the neutralization test, the sensitivity of the inhibition ELISA was 100% (111/111) for detection of anti‐PV1 antibody, 98.3% (118/120) for anti‐PV2, and 96.5% (82/85) for anti‐PV3, and the specificity was 93.1% (27/29), 100% (20/20), and 92.7% (51/55), respectively. Thus, the inhibition ELISA showed excellent potential as a seroepidemiologic tool in both vaccinated and naturally‐infected populations.

[Application of frozen stored various animal red blood cells for the virological and serological tests (author's transl)].

各種動物赤血球を用いておこなうウイルス学的, 血清学的試験に, 凍結保存赤血球を利用する目的で, 解凍赤血球の性能について試験をした結果, ヒト, ミドリザル, ヒツジ, モルモット, ニワトリおよび1日ビナ赤血球は, 非凍結赤血球と同様, 各種試験に使用できる成績を得た. すなわち, 上記各種動物凍結保存赤血球は, おのおの, 赤血球吸着, 赤血球凝集, 補体結合および免疫粘着反応に関与する特異的な性能を長期間保持していることを確認した.1. ヒトO型, ミドリザル, ヒツジおよびモルモット赤血球の血球沈渣を28w/v% glycerol, 3% mannitol, 0.65% NaCl液と等量混合し, 急速凍結, 液体窒素保存で長期に亘り保存できた.2. ニワトリおよび1日ビナ赤血球については, 血球沈渣を51.5w/v% dimethylsulfoxide (DMSO), 8% glucose, 1% fructose および0.3% EDTA-2Naからなる凍害防止液とを等量混合し, 緩速凍結後, -85℃で保存する方法が有効であった.3. 急速解凍後の脱グリセリンあるいは脱DMSOは十分量の16w/v% mannitol, 0.9% NaCl液で希釈する方法がよく, 等張液で洗浄し, 目的とする緩衝液におきかえた時点での回収率は, ヒト, サル, モルモットでは80%以上, ヒツジ, ニワトリでは65～80%, 1日ビナでは40～50%であった.4. 解凍赤血球は, 浸透圧に対する抵抗性が減弱する動物種もあったが, 少なくとも各種ウイルス学的, 血清学的反応に関与する表面構造は, 凍結, 解凍により障害を受けておらず, 非凍結の同種赤血球に匹敵する性能を示した.