抄録
In the past few years attention was focussed on heat resistant variant or mutant of polioviruses by several investigators (Melnick et al, Stanley et al, Dulbecco and Youngner). Along this line attempt was made to select heat resistant (t) mutant not only from a wild type such as MEF-1 t+ but from each parent type such as MEF-1 i t+ (a mutant resistant to inhibitors in normal bovine serum described by Takemori, Kitaoka et al) and MEF-1 i m t+ (a mutant giving small size plaque on HeLa cell-monolayer described by Takemori, Kitaoka et al). First the culture of a wild or a parental type especially type II poliovirus was heated at 50°C and variation in thermal resistance of individual infectious units was followed up by estimation of pfu/ml with exception of m-mutant stock which was titrated by cytopathic effect on HeLa cells in tubes, in 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes after exposure to the heat.
As a result surviving rate of wild type MEF-1 t+ fell down rapidly within 20 minutes after exposure to heat, that is, the heat sensitive virus was inactivated and approximately 0.3per cent of MEF-1 t+ was found to be heat resistant, phenotypically or genotypically, among a given virus population.
Herewith the selection of t-mutant was undertaken mainly according to Dulbecco's method, that is, by successive steps being propagation of survivors in subculture and heating at 50°C for 20 minutes one after another and if the surviving rates gave rise to 75per cent, two more subcultures were mabe through plaque passages or by the limiting dilution method to select possibly the thermostable clone.
The results so far obtained are as follows.
1) The t-mutant was selected at the 9th subculture of the wild type MEF-1 t+. Its surviving rate was more than 89per cent at 50°C for 20 minutes. It was found that progeny of different virus particles varied spontaneously in their rates of thermal inactivation.
2) The parental type MEF-1 i t+ was found to have 1.2 to 1.3 times much higher inactivation velosity constant than MEF-1 t+. The surviving rate of MEF-1 i t+ was 0.022per cent after heating at 50°C for 20 minutes. However, it increased and the incubation period became shorter giving cytopathic effect on HeLa cells at each generation one after another and the surviving rate of MEF-1 i t at the 4th and 5th generation was found to be more than 75per cent and the incubation period to give cytopathic effect became 3 days. It is worthy to note that progeny of MEF-1 i t retained its i-character in messing its plaque size on monolayer containing inhibitor in normal bovine serum and no differences was observed in i-character of the parent and mutant.
3) The difference between both TCID50 of the parental type MEF-1 m t+ before and after heating at 50°C for 20 minutes was 103.0 TCID50 by using HeLa cell tubes and the incubation period of the parental type giving cytopathic effect on HeLa was 6 days. However, the difference between both TCID50 of 2nd subculture of servival was 101.0 before and after heating. The incubation period of cytopathic effect on HeLa cells was shortened by subcultures and it became 2 days at the 3rd subculture. Thus MEF-1 m t was obtained without difficulty.
4) Furthermore, the back mutant MEF-1 m+t was selected from MEF-1 m t by serial passages in culture media containing bovine serum but its t-character was proven to be stable in spite of m-character back to m+.
5) The difference of TCID50 of MEF-1 m i t+ before and after heating was 102.5 but no difference was found at the 9th subculture, that is, complete t-mutant was obtained. Such t-mutant was confirmed to retain m- and i-properties in messing plaque size on monolayer containing inhibitors in bovine serum.
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