Co-occlusion and persistence of a baculovirus mutant lacking the polyhedrin gene.

A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. Coinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Autographa californica nuclear polyhedrosis virus (AcMNPV) and an AcMNPV mutant (Ac-E10) lacking the polyhedrin gene resulted in occlusion of both virus types within polyhedra. The amount of occluded Ac-E10 virions in progeny polyhedra populations during serial passage in Trichoplusia ni larvae was evaluated. Maintenance of the mutant in progeny polyhedra required polyhedra inocula containing equal numbers of the two virus types at a high dose. A significant reduction in occluded mutant nucleocapsids occurs with inoculum levels below a 100% lethal dose. At inoculum levels below a 30% lethal dose, the majority of fourth-instar larvae were infected with only one type of virus. The commercial application and ecological advantages of the co-occlusion process are discussed.

Rate of increase of Autographa californica nuclear polyhedrosis virus in Trichoplusia ni larvae determined by DNA:DNA hybridization.

The rate of increase and doubling time of the HOB clone of Autographa californica nuclear polyhedrosis virus (AcMNPV-HOB) in neonate Trichoplusia ni larvae was determined by measuring the increase in viral DNA through time following inoculation with average doses of 50 or 17,400 occlusion bodies per larva. Changes in total DNA and viral DNA through time were followed by fluorescence spectroscopy and quantitative slot-blot DNA:DNA hybridization, respectively. Total DNA content (i.e., larval DNA and viral DNA) of larvae infected with the intermediate dose lagged behind that of noninfected larvae 30 hr post-inoculation (p.i), reached a maximum at 51 hr p.i., and stayed constant thereafter. The total DNA content of larvae inoculated with the high dose lagged behind that of the control group from 18 hr p.i. and increased slowly until death of the larvae (ca. 48 hr p.i.). The amount of viral DNA in larvae inoculated with the intermediate dose increased exponentially between 15 and 42 hr p.i., reached a maximum at 48 hr p.i., and stayed constant until 68 hr p.i., by which time most larvae had died. The amount of viral DNA in larvae inoculated with the high dose did not increase exponentially; initially the rate of increase was the same as that for larvae inoculated with the intermediate dose but became progressively lower after 13 hr p.i. Calculations of the rate of increase for AcMNPV-HOB in neonate T. ni larvae inoculated with the intermediate dose and incubated at 29 degrees C resulted in a value of 0.264 hr-1 (doubling time: 2.63 hr).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Rate of increase and critical amount of nuclear polyhedrosis virus in lepidopterous larvae estimated from survival time assay data with a birth-death model.

A birth-death model developed for pathogens of vertebrates was used to estimate the in vivo rate of increase (alpha) and the doubling time (td) from survival time assay data. Host-pathogen combinations used in this study were two Autographa californica nuclear polyhedrosis virus isolates in Trichoplusia ni and Heliothis zea NPV in H. zea. The alpha's, estimated as he negative reciprocal of the slope of the linearly decreasing section of the plot of median survival times against the logarithm of inoculum concentration, were calculated in two ways. First, simple regression was used to fit a line through the linearly decreasing part using data points selected by eye; secondly, a three-phase segmented linear regression model was used and alpha was estimated from the slope of the middle segment. Estimates of alpha (and td) were 0.338 (2.05), 0.274 (2.53) and 0.243 h-1 (2.85 h) using the simple regression method, and 0.385 (1.80), 0.305 (2.27) and 0.223 h-1 (3.11 h) using the 3-phase segmented linear regression model for AcMNPV-1A, AcMNPV-HOB and HzSNPV, respectively. Although AcMNPV-HOB killed larvae faster (6 to 13 h) than AcMNPV-1A, it multiplied more slowly. Estimates for the critical number ranged from 4.8 x 10(9) to 4.5 x 10(14) genome copies for HzSNPV in H. zea larvae and AcMNPV-1A in T. ni larvae, respectively. The significance of the calculated critical numbers is discussed.