Nested PCR amplification of a Spodoptera litura nucleopolyhedrovirus (SpltNPV) lef-8 gene fragment was performed on egg masses and larvae of S. litura, and resulted in detection of a latent SpltNPV in 20.0% of “healthy” laboratory stock samples, and in 22.6% of samples collected at different periods and in remote places in Kagoshima Prefecture. The PCR product sequences showed 99% similarity to the published lef-8 sequence of SpltNPV, confirming that the amplification products were derived from SpltNPV. Further, cross-inoculation of laboratory stock S. litura larvae with Mythimna separata NPV (MyseNPV) activated the latent virus, which provoked a change in the virulence of MyseNPV samples collected from cadavers of S. litura. Our data suggest that caution should be taken when using S. litura as a host to produce heterologous NPVs.