A comparison of the adaptations of strains of Lymantria dispar multiple nucleopolyhedrovirus to hosts from spatially isolated populations.

The adaptation of pathogens to either their hosts or to environmental conditions is the focus of many current ecological studies. In this work we compared the ability of six spatially-distant Lymantria dispar (gypsy moth) multiple nucleopolyhedrovirus (LdMNPV) strains (three from eastern North America and three from central Asia) to induce acute infection in gypsy moth larvae. We also sequenced the complete genome of one Asian (LdMNPV-27/0) and one North American (LdMNPV-45/0) strain which were used for bioassay. We found that all of the North American virus strains, with the exception of one, demonstrated higher potency than the Asian virus strains, either in North American (Lymantria dispar) larvae or, in Asian (Lymantria dispar asiatica) larvae. Complete genome sequencing revealed two gene deletions in the LdMNPV-27/0 strain: the virus enhancin factor gene (vef-1) and the baculovirus repeated orf gene (bro-p). These deletions were not seen in the LdMNPV-45/0 strain nor in other American strains available in archiving systems. We also found deletions of the bro-e and bro-o genes in LdMNPV-45/0 strain but not in the LdMNPV-27/0 strain. The phylogenetic inference with an alignment of the 37 core gene nucleotide sequences revealed the close relationship of the LdMNPV-45/0 strain with other American strains accessed in GenBank (Ab-a624 and 5-6) while the LdMNPV-27/0 strain was clustered together with the LdMNPV-3054 strain (isolated in Spain) instead of predicted clustering with LdMNPV- 3029 (isolated in Asia). Our study demonstrated that first, different LdMNPV isolates from the same metapopulations of L. dispar exhibit little or no difference in the degree of virulence towards host larvae and second, that locality of host population is not an important driver of LdMNPV virulence. Virulence of LdMNPV is determined only by viral genetics. The genetic differences between North American and Central Asian virus strains are discussed.

The enhancin gene: One of the genetic determinants of population variation in baculoviral virulence.

It was established that the virulence of the North American baculovirus strain LdMNPV-45 is almost two orders of magnitude higher than the virulence of the Asian strain LdMNPV-27 and does not depend on the test host population (gypsy moth). The Asian strain carries deletions in bro-p and vef-1 genes (82 and 91%, respectively). In accordance with the published data, the product of the latter can greatly increase the virulence of the virus. This result indicates that the population polymorphism of the virulence of baculoviruses can be explained by the vef-1 gene deletion.

Interactions between an injected polydnavirus and per os baculovirus in gypsy moth larvae.

Larval gypsy moths, Lymantria dispar (Lepidoptera:Lymantriidae) were co-infected with the L. dispar nucleopolyhedrovirus (LdMNPV) and the Cotesia melanoscela (Hymenoptera:Braconidae) polydnavirus (CmeBV). CmeBV was given along with a parasitoid egg and calyx products in a stinging event, or in the form of an injection of calyx-derived extract. LdMNPV was delivered per os, integrated into artificial diet. Mortality from all sources was recorded over the subsequent three-week period. Neither parasitism nor injections of purified CmeBV with toxin had any effect on the amount of mortality caused by concurrent challenges with LdMNPV.

Physical map and polyhedrin gene sequence of Lymantria dispar nuclear polyhedrosis virus.

Restriction maps of the 166.6-kb genome of Lymantria dispar multiply-enveloped nuclear polyhedrosis virus (LdMNPV clone g) were constructed for BamHI, BglII, EcoRI, EcoRV, HindIII and KpnI, using cosmid pVK102 and pBluescript vectors. Southern hybridizations indicated that the LdMNPV genome contains five dispersed regions of intragenomic sequence homology. The polyhedrin gene of LdMNPV was located within BglII-E and the sequence of the 735-nucleotide (nt) coding region and 678 nt of flanking DNA was determined. A conserved 14-nt sequence, associated with transcriptional start points in other polyhedrins, was identified at 44 to 57 nt upstream from the start codon. The deduced polyhedrin amino acid (aa) sequence showed a high degree of homology with a previously determined protein sequence for LdMNPV polyhedrin (89%) and with deduced amino acid sequences for three other MNPV polyhedrins (74%). Optimal alignment of the four sequences indicated that LdMNPV polyhedrin possesses a single aa insertion at residue 4 and a single aa deletion at residue 164.

Field evaluation of a DNA hybridization assay for nuclear polyhedrosis virus in gypsy moth (Lepidoptera: Lymantriidae) larvae.

DNA hybridization assays were used to detect the presence of viral DNA in gypsy moth (Lymantria dispar L.) larvae collected weekly from high density populations or reared from field-collected egg masses. DNA was extracted from larvae, bound to nitrocellulose filters, and hybridized with digoxigenin-labeled L. dispar NPV (LdNPV) DNA probes. The virus incidence determined from DNA hybridization assays was compared with that determined with conventional microscopic examination of larvae for polyhedral inclusion bodies. Among neonates reared from field-collected egg masses, average mortality from LdNPV (15.4%) within 10 d after hatch was not significantly different from the percentage of extracts containing LdNPV DNA (14.8%) found among larvae frozen 5 d after hatch before any mortality occurred. Field-collected larvae were split into two groups: half were frozen immediately and probed for LdNPV DNA and the other half were reared on artificial diet. The proportion containing LdNPV DNA closely approximated the proportion that died within 6 d of collection, but the proportion that died within 13 d of collection was underestimated.

Genetic engineering of a Lymantria dispar nuclear polyhedrosis virus for expression of foreign genes.

A bacterial lacZ gene was inserted into an isolate of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV). The transfer vector was constructed by site-directed mutagenesis of the translation start site of the LdMNPV polyhedrin gene, within the BglII E fragment of the viral genome. A multiple cloning sequence was inserted at this start site and used for the insertion of the lacZ gene into the transfer plasmid. Liposome transfection was used to cotransfect L. dispar tissue culture cells with viral DNA and the transfer plasmid. Recombinant LdMNPV isolates were purified by isolation of plaques producing beta-galactosidase but not polyhedra. Restriction enzyme fragment profiles were used to determine the site of the lacZ gene insertion, and DNA sequencing of the 5' and 3' ends of the lacZ gene insert and the adjoining polyhedrin promoter and coding regions was performed to identify its precise location. Expression of the lacZ gene was examined by studying virus-induced protein using [35S]methionine pulse-labelling, SDS-PAGE fractionation and autoradiography. Expression of beta-galactosidase was examined in tissue culture cells using colorimetric assays. The maximum rate of beta-galactosidase production was approximately 50 international units (IU)/10(6) tissue culture cells/day between 3 and 4 days post-infection (p.i), and the peak total expression was 158 IU/10(6) cells 5 days p.i. beta-Galactosidase activity was first detected 48 h p.i. in haemolymph samples from fourth instar L. dispar larvae injected with 10(6) p.f.u. of virus. The peak beta-galactosidase activity in larval haemolymph samples was 1931 IU/ml of haemolymph at 11 days p.i., just prior to death.

A field release of genetically engineered gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (LdNPV).

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A beta-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for its presence using a colorimetric assay. In 1993, LdGEV-infected gypsy moths were released in a forested plot in Massachusetts to test for spread and persistence. A similar forested plot 2 km away served as a control. For 3 years (1993-1995), gypsy moths were established in the two plots in Massachusetts to serve as test and control populations. Each week, larvae were collected from both plots. These field-collected larvae were reared individually, checked for mortality, and then tested for the presence of beta-galactosidase. Other gypsy moth larvae were confined on LdGEV-contaminated foliage for 1 week and then treated as the field-collected larvae. The LdGEV was sought in bark, litter, and soil samples collected from each plot. To verify the presence of the LdGEV, polymerase chain reaction, slot blot DNA hybridization, and restriction enzyme analysis were also used on larval samples. Field-collected larvae infected with the engineered virus were recovered in the release plot in 1993, but not in subsequent years; no field-collected larvae from the control plot contained the engineered virus. Larvae confined on LdGEV-contaminated foliage were killed by the virus. No LdGEV was recovered from bark, litter, or soil samples from either of the plots.