Baculovirus Genome Research Articles

A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

Directional and Direct Cloning Strategy for High-Throughput Generation of Recombinant Baculoviruses

The baculovirus expression vector system (BEVS) has become one of the most widely used systems for routine protein expression. We have developed an improved strategy to clone foreign genes directionally and directly into the baculovirus genome vector via a one-step procedure to generate recombinant viruses in a week. In this work, we constructed a host strain Escherichia coli DH10BacHB1.1, which contains the modified baculovirus shuttle genome vector pHBMBacmid1.1 for the cloning vector. The treated PCR products of foreign genes were ligated with the Bsu36I-digested vector. Then Spodoptera frugiperda (Sf9) cells were transfected directly with the ligation mixture. Using this method, the DsRed fluorescence protein and mannanase genes have been cloned in the baculovirus genome and expressed in the Sf9 cells. This strategy not only provides a means for high-throughput construction of recombinant baculoviruses, but also offers an idea of constructing other large plasmids and DNA virus-based expression vectors.

Prospects for the Mosquito Baculovirus Cuninpv as a Tool for Mosquito Control

CuniNPV is a pathogen of Culex mosquitoes, vectors of West Nile virus and other forms of encephalitis. Successful development of CuniNPV requires an efficient production system and formulated product that incorporates magnesium, an essential component for transmission. It may be possible to develop mosquito baculoviruses as a new type of biopesticide by microencapsulating the virus and magnesium into formulations that would be effective regardless of the water quality. In addition, this new insight on transmission may facilitate the discovery and development of additional baculoviruses for the control of other important mosquito vectors. Biological mining of the CuniNPV genome and investigations to understand virus-mosquito interactions at the molecular level offer exciting possibilities for the development of novel mosquito control strategies and tools. Understanding the molecular mechanisms of infection will provide the opportunity to devise new control strategies, for example, compromising the defensive systems of the mosquito (proteases for the peritrophic matrix) or exploiting receptors used by the virus to specifically deliver toxins to mosquito larvae via the midgut. As additional baculovirus genomes become available, comparative genomics could lead to a more informed understanding of how the virus exploits its host as well as the factors responsible for the genus-specific host range of most known mosquito baculoviruses.

In vivo and in vitro analyses of a Bombyx mori nucleopolyhedrovirus mutant lacking functional vfgf

All lepidopteran baculovirus genomes sequenced to date encode a viral fibroblast growth factor homolog ( vfgf), suggesting that vfgf may play an important role in the infection cycle of lepidopteran baculoviruses. Here, we describe the characterization of a Bombyx mori nucleopolyhedrovirus (BmNPV) mutant lacking functional vfgf. We constructed a vfgf deletion mutant (BmFGFD) and characterized it in BmN cells and B. mori larvae. We observed that budded virus (BV) production was reduced in BmFGFD-infected BmN cells and B. mori larvae. The larval bioassays also revealed that deletion of vfgf did not reduce the infectivity; however, the mutant virus did take 20 h longer to kill B. mori larvae than wild-type BmNPV, when tested either by BV injection or by polyhedrin-inclusion body ingestion. These results suggest that BmNPV vfgf is involved in efficient virus production in BmN cells and B. mori larvae.

Sequence analysis of the Choristoneura occidentalis granulovirus genome

The genome of the Choristoneura occidentalis granulovirus (ChocGV) isolated from the western spruce budworm, Choristoneura occidentalis, was sequenced completely. It was 104,710 bp long, with a 67.3% A+T content and contained 116 potential open reading frames (ORFs) covering 88.4% of the genome. Of these, 29 ORFs were conserved in all fully sequenced baculovirus genomes, 30 were GV-specific, 53 were present in some nucleopolyhedroviruses (NPVs) and/or GVs, three were common to ChocGV and Choristoneura fumiferana GV (ChfuGV) and one was so far unique. To date, ChocGV is the only GV identified that contains a homologue of the apoptosis inhibitor protein P35/P49, present in some group I NPVs. It is also the first GV without a Xestia c-nigrum GV ORF 26 homologue. Five homologous regions (hrs)/repeat regions, lacking typical NPV hr palindromes were identified. ChocGV hrs were similar to each other but not to other GV hrs. A 1.8 kb repeat region with a high A+T content (81%) and multiple repeats of 21-210 bp was found between choc36 and 37. This area resembled the non-homologous region origin of DNA replication (non-hr ori) identified in Cryptophlebia leucotreta GV (CrleGV) and Cydia pomonella GV (CpGV). Based on the mean amino acid identities of homologous proteins, ChocGV was closest to fully sequenced genomes CpGV (52.3%) and CrleGV (52.1%). The closest amino acid identity was to individual ORFs from the partially sequenced ChfuGV genome (97.2% in 38 ORFs). Phylogenetic analysis placed ChocGV in a clade with CrleGV and CpGV.

Sequence Analysis and Organization of theNeodiprion abietisNucleopolyhedrovirus Genome

Of 30 baculovirus genomes that have been sequenced to date, the only nonlepidopteran baculoviruses include the dipteran Culex nigripalpus nucleopolyhedrovirus and two hymenopteran nucleopolyhedroviruses that infect the sawflies Neodiprion lecontei (NeleNPV) and Neodiprion sertifer (NeseNPV). This study provides a complete sequence and genome analysis of the nucleopolyhedrovirus that infects the balsam fir sawfly Neodiprion abietis (Hymenoptera, Symphyta, Diprionidae). The N. abietis nucleopolyhedrovirus (NeabNPV) is 84,264 bp in size, with a G+C content of 33.5%, and contains 93 predicted open reading frames (ORFs). Eleven predicted ORFs are unique to this baculovirus, 10 ORFs have a putative sequence homologue in the NeleNPV genome but not the NeseNPV genome, and 1 ORF (neab53) has a putative sequence homologue in the NeseNPV genome but not the NeleNPV genome. Specific repeat sequences are coincident with major genome rearrangements that distinguish NeabNPV and NeleNPV. Genes associated with these repeat regions encode a common amino acid motif, suggesting that they are a family of repeated contiguous gene clusters. Lepidopteran baculoviruses, similarly, have a family of repeated genes called the bro gene family. However, there is no significant sequence similarity between the NeabNPV and bro genes. Homologues of early-expressed genes such as ie-1 and lef-3 were absent in NeabNPV, as they are in the previously sequenced hymenopteran baculoviruses. Analyses of ORF upstream sequences identified potential temporally distinct genes on the basis of putative promoter elements.

Improving baculovirus transduction of mammalian cells by surface display of a RGD-motif

An RGD-containing peptide, comprising 23 amino acids from the foot-and-mouth disease virus (FMDV) VP1 protein was engineered into the envelope of Autographa californica nuclear polyhedrosis virus surface (AcNPV) using two different display strategies. The RGD-motif is a well-described tripeptide, that by binding to cell surface integrins facilitates virus entry into cells. This epitope was displayed, either by directly modifying the native major envelope protein gp64 of AcNPV, or by incorporating a second, modified version of gp64 onto the virus surface. Transduction efficiencies of four mammalian cell lines were compared by detecting the expression of the reporter gene green fluorescent protein (gfp), delivered by the baculovirus genome. Our results showed that insertion of the RGD-peptide into the envelope protein gp64 leads to enhanced specific uptake of baculoviral particles in mammalian cells, only when a combination of wild-type and mutant gp64 was present on the viral surface. Whenever the RGD-peptide was directly inserted into the native gp64, the overall amount of gp64 envelope protein was diminished, leading to decreased viral uptake.

On the classification and nomenclature of baculoviruses: A proposal for revision

Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran- and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).

Vaccines for Viral and Parasitic Diseases Produced with Baculovirus Vectors

The baculovirus–insect cell expression system is an approved system for the production of viral antigens with vaccine potential for humans and animals and has been used for production of subunit vaccines against parasitic diseases as well. Many candidate subunit vaccines have been expressed in this system and immunization commonly led to protective immunity against pathogen challenge. The first vaccines produced in insect cells for animal use are now on the market. This chapter deals with the tailoring of the baculovirus–insect cell expression system for vaccine production in terms of expression levels, integrity and immunogenicity of recombinant proteins, and baculovirus genome stability. Various expression strategies are discussed including chimeric, virus‐like particles, baculovirus display of foreign antigens on budded virions or in occlusion bodies, and specialized baculovirus vectors with mammalian promoters that express the antigen in the immunized individual. A historical overview shows the wide variety of viral (glyco)proteins that have successfully been expressed in this system for vaccine purposes. The potential of this expression system for antiparasite vaccines is illustrated. The combination of subunit vaccines and marker tests, both based on antigens expressed in insect cells, provides a powerful tool to combat disease and to monitor infectious agents.

Bombyx mori nucleopolyhedrovirus ORF79 encodes a 28-kDa structural protein of the ODV envelope

Open reading frame 79 of Bombyx mori nucleopolyhedrovirus (Bm79) is a conserved gene whose homologues have been identified in all 26 of the completely sequenced baculovirus genomes, including lepidopteran NPVs and GVs, hymenopteran NPVs, and a dipteran baculovirus. Northern blot analysis showed that the Bm79 transcript was about 850 nucleotides long and was initiated 12 h p.i. Temporal expression analysis revealed a 28-kDa protein, which was detected beginning 24 h p.i. using a polyclonal antibody against GST-Bm79 fusion protein. The 28-kDa protein was detected in the occlusion-derived virus envelope (ODV-E), but not in budded viruses. This observation was confirmed by observing ultrathin sections of polyhedra using immunoelectron microscopy. This demonstrated that the protein was present within the nuclei of cells. These results suggest that Bm79 is a functional gene that encodes a structural protein associated with the envelope of occlusion-derived virus (ODV).

An Anti-apoptosis Gene of the Bcl-2 Family from Marine Birnavirus Inhibiting Apoptosis of Insect Cells Infected with Baculovirus

VP5 is a 15-kDa nonstructural protein encoded by a small open reading frame in 5'-terminal of segment A of the Marine Birnavirus (MABV) (strainY-6) genome. Comparisons of the amino acid sequence of the VP5 with other Bcl-2 family member proteins indicated that the VP5 protein contains Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4, but without the transmembrane region. The VP5 gene from MABV was fused to enhancing green fluorescence protein (eGFP) gene and inserted into the baculovirus genome under the control of polyhedrin gene promoter, and then was highly expressed in insect cells. The expressed VP5 was capable of enhancing insect cell viability, prevented membrane blebbing and delayed DNA internucleosomal cleavage when cells were infected with the recombinant virus. The results suggested that the VP5 of MABV is a novel anti-apoptosis gene, which could regulate the cell apoptosis-off system.

Functional analysis of evolutionary conserved clustering of bZIP binding sites in the baculovirus homologous regions ( hrs) suggests a cooperativity between host and viral transcription factors

The genome of the Autographa californica Multinucleocapsid Polyhedrosis Virus ( AcMNPV) contains nine interspersed homologous regions ( hrs) that function as potent enhancer sequences when linked in cis to either viral or heterologous RNA polymerase II-dependent promoters. Their activity is strongly increased by the binding of the major immediate early viral transregulator IE1 on 28-mer palindromic sites present in hrs. We show that hrs of AcMNPV additionally carry, in the interpalindromic sequences, a large number of cAMP response elements (CRE) and TPA response elements (TRE), known to bind ubiquitous cellular transcription factors of the bZIP family. Moreover, these clusters of CRE and TRE motifs are concentrated in hrs. Analysis of the 25 baculovirus genomes sequenced so far reveals that these motifs are evolutionary conserved in Lepidoptera NPVs, suggesting a functional role in the hr enhancer function. Consistently, EMSA experiments indicate that CRE and on a lesser extent TRE sites specifically bind insect host factors. Moreover, reporter assays reveal that these CRE sites have an additive stimulatory effect on RNAPol II-dependent transcription in Sf9 cells and are potentially able to synergize with the IE1-binding palindrome.

P300/CBP-associated Factor Drives DEK into Interchromatin Granule Clusters

DEK is a mammalian protein that has been implicated in the pathogenesis of autoimmune diseases and cancer, including acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. In addition, DEK appears to participate in multiple cellular processes, including transcriptional repression, mRNA processing, and chromatin remodeling. Sub-nuclear distribution of this protein, with the attendant functional ramifications, has remained a controversial topic. Here we report that DEK undergoes acetylation in vivo at lysine residues within the first 70 N-terminal amino acids. Acetylation of DEK decreases its affinity for DNA elements within the promoter, which is consistent with the involvement of DEK in transcriptional repression. Furthermore, deacetylase inhibition results in accumulation of DEK within interchromatin granule clusters (IGCs), sub-nuclear structures that contain RNA processing factors. Overexpression of P/CAF acetylase drives DEK into IGCs, and addition of a newly developed, synthetic, cell-permeable P/CAF inhibitor blocks this movement. To our knowledge, this is the first reported example of acetylation playing a direct role in relocation of a protein to IGCs, and this may explain how DEK can function in multiple pathways that take place in distinct sub-nuclear compartments. These findings also suggest that DEK-associated malignancies and autoimmune diseases might be amenable to treatment with agents that alter acetylation.

Improvements to the throughput of recombinant protein expression in the baculovirus/insect cell system

Recombinant baculoviruses have proved to be a very useful means to express many proteins over the last 20 years. Since their introduction, there have been a number of significant improvements that have simplified and speeded up the construction of baculoviruses. One of the most commonly used methods relies upon recombination with the baculovirus genome maintained in Escherichia coli. In this paper, we report the conversion of nearly all the steps in this process including the expression testing and purification to a multi-well plate format. This enables a significant increase in the number of constructs that can be processed in a shorter period of time and an order of magnitude increase in the number of expression conditions that can be analysed. A key step in our process is that the transfection is done in suspension rather than adherent cells, which gives a much higher virus titre than in the standard methods.

Analysis of the Choristoneura fumiferana nucleopolyhedrovirus genome

The double-stranded DNA genome of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) was sequenced and analysed in the context of other group I nucleopolyhedroviruses (NPVs). The genome consists of 129,593 bp with a G + C content of 50.1 mol%. A total of 146 open reading frames (ORFs) of greater than 150 bp, and with no or minimal overlap were identified. In addition, five homologous regions were identified containing 7-10 repeats of a 36 bp imperfect palindromic core. Comparison with other completely sequenced baculovirus genomes revealed that 139 of the CfMNPV ORFs have homologues in at least one other baculovirus and seven ORFs are unique to CfMNPV. Of the 117 CfMNPV ORFs common to all group I NPVs, 12 are exclusive to group I NPVs. Overall, CfMNPV is most similar to Orgyia pseudotsugata MNPV based on gene content, arrangement and overall amino acid identity. Unlike other group I baculoviruses, however, CfMNPV encodes a viral enhancing factor (vef) and has two copies of p26.

Gene organization and sequencing of the Choristoneura fumiferana defective nucleopolyhedrovirus genome

Two distinct nucleopolyhedrovirus species of the eastern spruce budworm, Choristoneura fumiferana, exist in a symbiont-like relationship. C. fumiferana defective nucleopolyhedrovirus (CfDEFNPV) only infects C. fumiferana larvae per os in the presence of C. fumiferana nucleopolyhedrovirus Ireland strain (CfMNPV), but is infective when injected into the haemolymph. CfDEFNPV synergizes CfMNPV in per os infections and CfMNPV is always the predominant progeny. This study was undertaken to report the genomic makeup and organization of CfDEFNPV in an attempt to identify its defect and understand its synergistic role. The genome was mapped, sequenced, characterized and compared to other baculoviruses. The CfDEFNPV genome was 131,160 nt long with 149 putative open reading frames (ORFs) and a G + C content of 45.8 mol%. Homologues of all 62 conserved lepidopteran baculovirus genes were found including those implicated in per os infectivity, p74, per os infectivity factor (pif) and pif-2. Although no obvious deletions were observed to explain the defect, two ORFs, Cfdef79 and Cfdef99 (inhibitor of apoptosis-4), contained potential deletions. Cfdef50 (late expression factor-10)/Cfdef51 (vp1054) and Cfdef76/Cfdef77 (telokin-like protein) had large overlaps and a potential homologue to ac105/he65 was split. Four baculovirus repeat ORFs were present, as were two unique genes, but no enhancins were identified. CfDEFNPV contained 13 homologous regions, each with one to five palindromes. Comparison with fully sequenced baculovirus genomes identified CfDEFNPV as a group I NPV with the closest average amino acid identity to Epiphyas postvittana NPV, followed by Orgyia pseudotsugata MNPV and CfMNPV, with its closest matches being to individual Anticarsia gemmatalis MNPV gene sequences.

CYP2K6 from zebrafish ( Danio rerio): Cloning, mapping, developmental/tissue expression, and aflatoxin B1 activation by baculovirus expressed enzyme

A full-length zebrafish ( Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5′-untranslated region (5′-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3′-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5′-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (ω-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1–GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry–mass spectrometry (MS–MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.

Gross Pathology of SpliNPVs and Alterations in Spodoptera littoralis Boisd. (Lepidoptera:Noctuidae) Morphology Due to Baculoviral Infection*

Baculoviruses are invertebrate-specific pathogens and baculoviral infections cause alterations in the physiology, metabolism and morphology of insects. It is important to recognize these physiological and symptomatologic changes to understand baculovirus infection cycle and biology. For this reason, in our study Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) larvae were inoculated with the SpliNPV to doses of 3000 and 20,000 OBs for third instars and to concentrations of 10 6 and 3x 10 6 OBs/ml for neonates and the alterations due to NPV infection in larvae were then examined. Bioassays carried out with the third instar S. littoralis larvae revealed that no symptoms were detected during the first three days post-inoculation due to SpliNPV infection. Approximately, in the 4th day, infected larvae began to respond much more slowly than healthy larvae. This symptom was followed by whitening and slimming of cuticula, failure in molting, swelling of the body, diarrhoea, climbing to high places and hanging, liquefaction of the body and death. Infected neonates exhibited limited symptoms of distension and the failure of molting. The larvae died in approximately 8-8.5 days for third instars and in approximately 3-3.5 days for neonates due to baculoviral infection for the both doses and concentrations. On the other hand, different doses or concentrations did not cause an alteration in the occurence time of symptoms. To associate the NPV biology with the genes involved in baculovirus genome and understand their life cycle will improve their efficacy as biopesticides and help to the effective use of baculoviruses.

Complete comparative genomic analysis of two field isolates of Mamestra configurata nucleopolyhedrovirus-A

A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant 90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was 99.5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the v90/4 genome has 521 point mutations and numerous deletions and insertions when compared to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region between hr1 and orf31 shows the greatest divergence between the two genomes. This region contains three bro genes, which are among the most variable baculovirus genes. These results, together with other published data, suggest that bro genes may influence baculovirus genome diversity and may be involved in recombination between baculovirus genomes. Many ambiguous residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms. Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the 90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent approximately 25 % of the genotypes in the field isolate. These results provide evidence of extensive sequence variation among the individual genomes comprising a natural baculovirus outbreak in a continuous host population.

Sequence analysis of the genome of the Neodiprion sertifer nucleopolyhedrovirus.

The genome of the Neodiprion sertifer nucleopolyhedrovirus (NeseNPV), which infects the European pine sawfly, N. sertifer (Hymenoptera: Diprionidae), was sequenced and analyzed. The genome was 86,462 bp in size. The C+G content of 34% was lower than that of the majority of baculoviruses. A total of 90 methionine-initiated open reading frames (ORFs) with more than 50 amino acids and minimal overlapping were found. From those, 43 ORFs were homologous to other baculovirus ORFs, and 29 of these were from the 30 conserved core genes among all baculoviruses. A NeseNPV homolog to the ld130 gene, which is present in all other baculovirus genomes sequenced to date, could not be identified. Six NeseNPV ORFs were similar to non-baculovirus-related genes, one of which was a trypsin-like gene. Only one iap gene, containing a single BIR motif and a RING finger, was found in NeseNPV. Two NeseNPV ORFs (nese18 and nese19) were duplicates transcribed in opposite orientations from each other. NeseNPV did not have an AcMNPV ORF 2 homolog characterized as the baculovirus repeat ORF (bro). Six homologous regions (hrs) were located within the NeseNPV genome, each containing small palindromes embedded within direct repeats. A phylogenetic analysis was done to root the tree based upon the sequences of DNA polymerase genes of NeseNPV, 23 other baculoviruses, and other phyla. Baculovirus phylogeny was then constructed with 29 conserved genes from 24 baculovirus genomes. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was the most distantly related baculovirus, branching to the hymenopteran NeseNPV and the lepidopteran nucleopolyhedroviruses and granuloviruses.