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Recombinant baculoviruses obtained by coinfection ofinsect cells with Autographa californica and Bombyx mori nuclear polyhedrosis viruses (AcNPV and BmNPV, respectively) possess a wider in vitro host range than either parent virus. To localize the DNA sequences responsible for this species specificity, we used a two-step method of production and selection of recombinant viruses with altered specificity.

Autographa californica nucleopolyhedrovirus (Ac-MNPV) does not replicate in Bombyx mori cells (Bm5, BmN). We have shown previously that when a short DNA sequence within AcMNPV ORF95, which encodes the viral helicase P143, is replaced with the collinear region of B. mori nucleopolyhedrovirus (BmNPV), AcMNPV gains the ability to replicate in Bm5 cells. To determine the mutational events in the p143 gene required to allow AcMNPV replication in B. mori cells, AcMNPV recombinants produced in Sf9 cells were screened in vivo in B. mori larvae, which are more permissive to baculovirus infection than B. mori cell lines. Eight combinations of mutations were tested and characterization of viral DNA extracted from dead larvae showed that amino acid changes at position 564 and 577 are required to kill B. mori larvae.

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