A newly cloned Helicoverpa armigera nucleopolyhedrovirus (HearNPV) from Kenya, HearNPV-NNg1, has a higher insecticidal activity than HearNPV-G4, which also exhibits lower insecticidal activity than HearNPV-C1. In the search for genes and/or nucleotide sequences that might be involved in the observed virulence differences among Helicoverpa spp. NPVs, the entire genome of NNg1 was sequenced and compared with previously sequenced genomes of G4, C1 and Helicoverpa zea single-nucleocapsid NPV (Hz). The NNg1 genome was 132,425 bp in length, with a total of 143 putative open reading frames (ORFs), and shared high levels of overall amino acid and nucleotide sequence identities with G4, C1 and Hz. Three NNg1 ORFs, ORF5, ORF100 and ORF124, which were shared with C1, were absent in G4 and Hz, while NNg1 and C1 were missing a homologue of G4/Hz ORF5. Another three ORFs, ORF60 (bro-b), ORF119 and ORF120, and one direct repeat sequence (dr) were unique to NNg1. Relative to the overall nucleotide sequence identity, lower sequence identities were observed between NNg1 hrs and the homologous hrs in the other three Helicoverpa spp. NPVs, despite containing the same number of hrs located at essentially the same positions on the genomes. Differences were also observed between NNg1 and each of the other three Helicoverpa spp. NPVs in the diversity of bro genes encoded on the genomes. These results indicate several putative genes and nucleotide sequences that may be responsible for the virulence differences observed among Helicoverpa spp., yet the specific genes and/or nucleotide sequences responsible have not been identified.

A gene encoding a putative DNA polymerase (pol) of Bombyx mori nuclear polyhedrosis virus (BmSNPV) was cloned and sequenced. The gene included an open reading frame (ORF) encoding a polypeptide of 988 amino acids with a predicted molecular mass of 114.65 kDa. The deduced amino acid sequence of the BmSNPV pol ORF showed an overall identity of 96 and 45% to those of the Autographa californica NPV (AcMNPV) pol ORF and the Lymantria dispar NPV pol ORF, respectively, and contained sequences conserved in a variety of eukaryotic and viral replicative DNA polymerases. The BmSNPV pol lacked a canonical TATAA element but contained a G+C-rich sequence in the transcriptional initiation region. Analyses by Northern blot hybridization, RNase protection assay, primer extension, and 3' and 5' RACE (rapid amplification of cDNA ends) showed that at least seven different transcripts of approximately 3.1 kb that shared a common 3' end were expressed from the BmSNPV pol. The expression of these transcripts from BmSNPV pol was regulated differentially during virus infection. Transcription of five of the seven species initiated in the close vicinity of and within the motif 5'-GCGTGCT-3'. One transcript placed its initiation site within the motif 5'-AGAGCGT-3' and the remaining one within the motif 5'-GGCGGTGG-3'. The motifs 5'-GCGTGCT-3' and 5'-AGAGCGT-3' have been identified in pol and other genes of AcMNPV as conserved sequences containing transcriptional initiation sites, whereas the motif 5'-GGCGGTGG-3', which is arranged as a direct repeat in BmSNPV pol but not in AcMNPV pol, has not been defined as the sequence responsible for transcriptional initiation sites. The BmSNPV pol transcripts were detectable at 2 hr postinfection (p.i.), peaked at 10 hr p.i., and declined to a low level by 18 hr p.i. The expression of BmSNPV pol was not inhibited but delayed dramatically by the protein synthesis inhibitor cycloheximide upon treatment of infected cells, whereas aphidicolin, an inhibitor of DNA polymerase, inhibited BmSNPV pol transcription. These results suggest a complicated and unique mechanism for the regulation of BmSNPV pol expression.

The whole-genome sequence of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) was analysed. The entire nucleotide sequence of the HycuNPV genome was 132 959 bp long, with a G+C content of 45?1 mol%. A total of 148 open reading frames (ORFs) consisting of more than 50 aa were encoded by the genome. HycuNPV shares more than 122 ORFs with other lepidopteran group I NPVs, including Autographa californica MNPV, Bombyx mori NPV, Choristoneura fumiferana MNPV (CfMNPV), Choristoneura fumiferana defective NPV, Epiphyas postvittana MNPV and Orgyia pseudotsugata MNPV (OpMNPV). Six ORFs are identified as being unique to HycuNPV. Most of the HycuNPV ORFs showed higher similarity to CfMNPV and OpMNPV ORFs than to those of the other group I NPVs. HycuNPV encodes two conotoxin-like homologues (ctls), which are observed only in OpMNPV in group I NPVs. HycuNPV encodes three inhibitors of apoptosis (iaps), hycu-iap-1, hycu-iap-2 and hycu-iap-3, a feature that it shares only with CfMNPV. In addition, six homologous regions (hrs) are identified in the HycuNPV genome. These hrs are located in regions similar to those of the OpMNPV hrs, but different from most of the CfMNPV hrs. Based on the close phylogenetic relationship and conservation of group I NPV-specific genes, such as gp64, ie-2 and ptp-1, it is concluded that HycuNPV belongs to the group I NPVs and is most similar to CfMNPV or OpMNPV.

 Abstract-Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is highly pathogenic to the cotton bollworm, H. armigera. There have been several attempts to develop in vitro production of this virus, including the selection of high producing cell line. In order to evaluate the potential of in vitro methods of producing this virus on a large-scale, the virus was propagated in the H. armigera (HAPO2) and H. zea (HZ-AM1) cell lines and the correlation between the few polyhedra (FP) phenotype of HearNPV and the mutation in the polyhedrin (polh) and fp25k genes was analyzed. The many polyhedra (MP) phenotype was the predominant population of HearNPV for 3 passages in HZ-AM1 culture. The production of occlusion bodies (OBs) decreased with increasing passage number. The phenotypic observations on the HearNPV FP variants when this virus is serially passaged in HZ-AM1 cells suggest genomic changes occurring in the viral genes. A single point mutation resulting in an amino acid change was identified in the fp25kgene. This mutation could be correlated with the appearance of the FP phenotype with no relation to the polh gene. In contrast, serial passage in HAPO2 cells showed a slower accumulation of FP variants when compared to the use of HZ-AM1 cell line and no mutation was found in the fp25k gene. Therefore, HAPO2 cell line is a productive new cell line that will be useful for the scale-up process of HearNPV bioinsecticides.

A continuous cell line, designated KU-SENL-1 (SENL1), has been established from minced neonate larvae of Spodoptera exigua (Lepidoptera: Noctuidae) treated with collagenase. The primary culture was maintained in TC100 medium supplemented with 10% Fetal Bovine Serum (FBS), 3% Helicoverpa armigera hemolymph and incubated at 27°C. This continuous cell line was cultured in TC100 medium supplemented with 10% FBS and subcultured at 5day intervals. The cell line consisted of a mixture of two cell types, epithelial-like cells and spindle-shaped cells, both of which grown as attached monolayers. The population doubling time of this new cell line during the logarithmic phase of growth was 45h. RAPD and DAF analyses confirmed that the origination of the SENL1 cell line was S. exigua. The susceptibility of this cell line to the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was high and by 3 days postinfection (pi) greater than 90% of the cells contained Occlusion Bodies (OBs) or were greatly hypertrophied, indicating they were infected. This cell line was highly effective for Budded Viruses (BV) titration of the AcMNPV. Therefore, the SENL1 cell line will be a valuable new tool for biological characterization of AcMNPV in cell culture and also for protein expression using the baculovirus-insect cell expression vector system.  Index Terms-Spodoptera exigua, Autographa californica, nucleopolyhedrovirus, insect cell culture, plaque assay II. MATERIALS AND METHODS A. Primary Culture and Subculture A laboratory colony of S. exigua was reared on an artificial diet [14] and allowed to grow to the pupal stage. At adult emergence, 10 females and an equal number of