Temperature-sensitive (ts) mutants of Bombyx mori nucleopolyhedrovirus (BmNPV), which exhibited no polyhedra formation at the non-permissive temperature of 33 degrees C, were produced using the base analogue 5-bromodeoxyuridine. A unique ts mutant, designated ts-S1, was characterized in terms of gene mutation and virulence in cultured cells and silkworm larvae. Mutant-infected BmN4 cells at 33 degrees C showed normal viral DNA synthesis but defective budded virus production and polyhedrin synthesis, suggesting the absence of late and very late gene expression. Silkworm larvae were injected with ts-S1 and reared at 33.5 degrees C. At 7 days post-injection, none of the larvae exhibited nucleopolyhedrosis but some possessed viral DNA, detected by PCR using virus-specific primers. Continued rearing of the larvae at a permissive temperature of 25 degrees C showed that, while most developed into normal adults, some developed nucleopolyhedrosis, indicating that the former larvae had aborted the virus infection during the course of rearing at 33.5 degrees C. No viral DNA was detected in the adults. Marker rescue tests to identify the lesion involved in the ts phenotype of ts-S1, and nucleotide sequencing of the identified genome region, showed a single nucleotide mutation of a putative RNA polymerase gene, late expression factor-8 (lef-8). These results indicate that lef-8 is essential for BmNPV replication in vitro and in vivo.


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