Lymantria dispar nucleopolyhedrovirus hrf-1 expands the larval host range of Autographa californica nucleopolyhedrovirus

Abstract

The gypsy moth (Lymantria dispar) is nonpermissive for Autographa californica nucleopolyhedrovirus (AcNPV) infection. We previously isolated a gene, host range factor 1 (hrf-1), from L. dispar nucleopolyhedrovirus that promotes AcNPV replication in Ld652Y cells, a nonpermissive L. dispar cell line (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996). In the present study, we investigated the ability of hrf-1 to alter the larval host range of AcNPV. Bioassays using recombinant AcNPV bearing hrf-1 were conducted with insect larvae by use of oral infection. AcNPV bearing hrf-1 was infectious for neonate L. dispar larvae, with a 50% lethal concentration of 1.2 x 10(5) polyhedral inclusion bodies/ml of diet, which is similar to that of wild-type AcNPV for permissive hosts. AcNPV can kill neonate L. dispar larvae at high doses, but it does not kill third-instar larvae. However, electron microscopy studies of AcNPV-inoculated third-instar larvae revealed virus replication in the midgut cells. PCR analyses indicated that the virus was AcNPV. These results suggest that the block for AcNPV infection of L. dispar larvae is its inability to spread systematically from primary infection sites in the midgut epithelium and that this barrier is leaky in neonates. hrf-1 allows AcNPV to overcome this barrier. AcNPV recombinants bearing hrf-1 were also significantly more infectious for Helicoverpa zea, a resistant species, suggesting that the blocks for AcNPV infection of L. dispar and H. zea larvae may be similar.

FIG. 1

(A) Sequence comparisons of various baculovirus polyhedrin gene sequences in the region used for PCR primers and restriction analysis. Abbreviations: Bm, Bombyx mori; Ag, Anticarsia gemmatalis; Op, Orgyia pseudotsugata; Mb, M. brassicae; Pf, Panolis flammea; Se, S. exigua; Sf, S. frugiperda; Hz, H. zea. Primer pairs F (5′-AGAACGCTAAGCGCAAGAAGCA-3′) and R (5′-GGCTTGTAGAAGTTCTCCCA-3′) are based on the AcNPV polyhedrin sequence and map to nucleotides 4603 to 4625 and 5136 to 5117 of the AcNPV genome, respectively (1). Locations of diagnostic HindIII and BamHI sites and sizes of predicted PCR product and restriction fragments are indicated. (For the polh sequence, see references and 37). (B) PCR products amplified by using primer pairs specific to the AcNPV polyhedrin gene. AcNPV DNA (lane 1), DNA isolated from OV from moribund AcNPV-inoculated L. dispar larvae (lane 2), LdNPV DNA (lane 3), and S. exigua NPV DNA (lane 4) were amplified. The amplified products from lanes 1 to 4 were digested with HindIII (lanes 5 to 8, respectively). Predicted amplification and digestion products are indicated by arrows to the right of the panel. The size markers on the left are given in kilobase pairs. (C) PCR products amplified by using the AcNPV polh primer pair from DNA isolated from third-instar L. dispar midgut tissues or AcNPV DNA as the template. AcNPV-infected larvae at 6, 12, 24, 48, and 60 h p.i. (lanes 1 to 5, respectively), AcNPV DNA (lane 6), or mock-infected larvae (lane 7) were used. Amplified products from 6 and 60 h p.i. were digested with HindIII (lanes 8 and 9, respectively) and compared to the HindIII-digested amplification product from AcNPV DNA (lane 10). The predicted 354- and 180-bp restriction fragments are indicated with arrows. Size markers on the left are given in base pairs.

FIG. 1

(A) Sequence comparisons of various baculovirus polyhedrin gene sequences in the region used for PCR primers and restriction analysis. Abbreviations: Bm, Bombyx mori; Ag, Anticarsia gemmatalis; Op, Orgyia pseudotsugata; Mb, M. brassicae; Pf, Panolis flammea; Se, S. exigua; Sf, S. frugiperda; Hz, H. zea. Primer pairs F (5′-AGAACGCTAAGCGCAAGAAGCA-3′) and R (5′-GGCTTGTAGAAGTTCTCCCA-3′) are based on the AcNPV polyhedrin sequence and map to nucleotides 4603 to 4625 and 5136 to 5117 of the AcNPV genome, respectively (1). Locations of diagnostic HindIII and BamHI sites and sizes of predicted PCR product and restriction fragments are indicated. (For the polh sequence, see references and 37). (B) PCR products amplified by using primer pairs specific to the AcNPV polyhedrin gene. AcNPV DNA (lane 1), DNA isolated from OV from moribund AcNPV-inoculated L. dispar larvae (lane 2), LdNPV DNA (lane 3), and S. exigua NPV DNA (lane 4) were amplified. The amplified products from lanes 1 to 4 were digested with HindIII (lanes 5 to 8, respectively). Predicted amplification and digestion products are indicated by arrows to the right of the panel. The size markers on the left are given in kilobase pairs. (C) PCR products amplified by using the AcNPV polh primer pair from DNA isolated from third-instar L. dispar midgut tissues or AcNPV DNA as the template. AcNPV-infected larvae at 6, 12, 24, 48, and 60 h p.i. (lanes 1 to 5, respectively), AcNPV DNA (lane 6), or mock-infected larvae (lane 7) were used. Amplified products from 6 and 60 h p.i. were digested with HindIII (lanes 8 and 9, respectively) and compared to the HindIII-digested amplification product from AcNPV DNA (lane 10). The predicted 354- and 180-bp restriction fragments are indicated with arrows. Size markers on the left are given in base pairs.

FIG. 2

Electron micrographs of AcNPV-infected third-instar L. dispar larval midgut cells at 48 h p.i. (A) Nucleocapsids assembling around virogenic stroma in cell nucleus. Magnification, ×19,000. Bar, 0.76 μm. (B) A nucleocapsid budding through the nuclear membrane. Magnification, ×36,000. Bar, 0.4 μm. Abbreviations: C, cytoplasm; CS, capsid sheath; NE, nuclear envelope; NC, nucleocapsid; NM, nuclear membrane; Nu, nucleus; VS, virogenic stroma.