Genome Sequencing and Organization of Three Geographically Different Isolates of Nucleopolyhedrovirus from the Gypsy Moth Reveal Significant Genomic Differences.

Background: The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is a worldwide pest of trees and forests. Lymantria dispar nucleopolyhedrovirus (LdMNPV) belongs to the Baculoviridae family and is an insect virus specific to gypsy moth larvae. In this study, we describe the complete genome sequences of three geographically diverse isolates, H2 (China), J2 (Japan), and T3 (Turkey), of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Methods: The genomes of isolates H2, J2, and T3 were subjected to shotgun pyrosequencing using Roche 454 FLX and assembled using Roche GS De Novo Assembler. Comparative analysis of all isolates was performed using bioinformatics methods. Results: The genomes of LdMNPV-H2, J2, and T3 were 164,746, 162,249, and 162,614 bp in size, had GC content of 57.25%, 57.30%, and 57.46%, and contained 162, 165, and 164 putative open reading frames (ORFs ≥ 150 nt), respectively. Comparison between the reference genome LdMNPV-5/6 (AF081810) and the genomes of LdMNPV-H2, J2, and T3 revealed differences in gene content. Compared with LdMNPV-5/6, ORF5, 6, 8, 10, 31, and 67 were absent in LdMNPV-H2, ORF5, 13, and 66 were absent in LdMNPV-J2, and ORF10, 13, 31, and 67 were absent in LdMNPV-T3. In addition, the gene encoding the mucin-like protein (ORF4) was split into two parts in isolates H2 and T3 and designated ORF4a and ORF4b. Phylogenetic analysis grouped isolates H2 and J2 in a different cluster than isolate T3, which is more closely related to the Turkish and Polish isolates. In addition, H2 was found to be closely related to a South Korean LdMNPV isolate. Conclusion: This study provided a more detailed overview of the relationships between different geographic LdMNPV isolates. The results showed remarkable differences between groups at the genome level.

Genome Characteristics of the Cyclophragma Undans Nucleopolyhedrovirus: A Distinct Species in Group I of Alphabaculovirus.

The Cyclophragma undans nucleopolyhedrovirus (CyunNPV), a potential pest control agent, was isolated from Cyclophragma undans (Lepidoptera: Lasiocampidae), an important forest pest. In the present study, we performed detailed genome analysis of CyunNPV and compared its genome to those of other Group I alphabaculoviruses. Sequencing of the CyunNPV genome using the Roche 454 sequencing system generated 142,900 bp with a G + C content of 45%. Genome analysis predicted a total of 147 hypothetical open reading frames comprising 38 baculoviral core genes, 24 lepidopteran baculovirus conserved genes, nine Group I Alphabaculovirus conserved genes, 71 common genes, and five genes that are unique to CyunNPV. In addition, the genome contains 13 homologous repeated sequences (hrs). Phylogenetic analysis groups CyunNPV under a distinct branch within clade "a" of Group I in the genus Alphabaculovirus. Unlike other members of Group I, CyunNPV harbors only nine of the 11 genes previously determined to be specific to Group I viruses. Furthermore, the CyunNPV lacks the tyrosine phosphatase gene and the ac30 gene. The CyunNPV F-like protein contains two insertions of continuous polar amino acids, one at the conventional fusion peptide and a second insertion at the pre-transmembrane domain. The insertions are likely to affect the fusion function and suggest an evolutionary process that led to inactivation of the F-like protein. The above findings imply that CyunNPV is a distinct species under Group I Alphabaculovirus.

Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248).

Amsacta moorei entomopoxvirus (AMEV) infects certain lepidopteran and orthopteran insects and is the most studied member of the genus Betaentomopoxvirus. It has been considered as a potential vector for gene therapy, a vector to express exogenous proteins and a biological control agent. One of its open reading frames, amv248, encodes a putative glycosyltransferase and is the only known attachment protein conserved in AMEV and chordopoxviruses. The ORF was successfully expressed and the protein was shown to bind soluble heparin, both in silico and in vitro. Our results also showed that, while viral infection was inhibited by soluble glycosaminoglycans (GAGs), GAG-deficient cells were more resistant to the virus. Finally, we revealed that amv248 encodes an active heparin-binding glycosyltransferase which is likely to have a key role in the initiation of infection by AMEV.

Transcriptional analysis of the putative glycosyltransferase gene (amv248) of the Amsacta moorei entomopoxvirus.

Amsacta moorei entomopoxvirus (AMEV), the most studied member of the genus Betaentomopoxvirus, was initially isolated from Red Hairy caterpillar larvae, Amsacta moorei. According to genome sequence and previous studies it was shown that amv248 encodes a putative glycosyltransferase that is the only conserved attachment protein in betaentomopoxviruses. Transcriptional analysis of the amv248 gene by RT-PCR and qPCR showed that transcription starts at 6h post infection (hpi). Also, transcription was not affected by a DNA replication inhibitor but was severely curtailed by a protein synthesis inhibitor. These results indicate that amv248 belongs to the intermediate class of gene expression. 5' and 3' untranslated regions analysis revealed that transcription initiates at position -126 relative to the translational start site, and ends between 50 and 83 bases after the stop codon. To narrow down the size and location of the gene's promoter, the upstream region as well as several different sized deletions thereof were generated and cloned upstream of a luciferase reporter gene. The constructs were used to measure the Firefly and Renilla luciferase activities in dual assays. The results showed that luciferase activity decreased when bases -198 to -235 of amv248 upstream region were missing. Sequence analysis among the intermediate gene promoters of AMEV showed that TTTAT(T/A)TT(T/A)2TTA is possibly a common motif, however, further investigations are needed to confirm this conclusion.

Construction and Rescue of a Functional Synthetic Baculovirus.

Synthetic viruses provide a powerful platform to delve deeper into the nature and function of viruses as well as to engineer viruses with novel properties. So far, most synthetic viruses have been RNA viruses (<30 kb) and small DNA viruses, such as bacteriophage phiX174. Baculoviruses contain a large circular dsDNA genome of 80-180 kb and have been used as biocontrol agents and protein expression vectors. Here, we report on the first synthesis of a baculovirus based on the type species Autographa californica nucleopolyhedrovirus, AcMNPV, by a combination of PCR and transformation-associated recombination in yeast. The synthetic genome, designated AcMNPV-WIV-Syn1, is 145 299 bp comprising the complete genome of AcMNPV except for the hr4a locus that was replaced with an ∼11.5 kb cassette of bacterial and yeast artificial chromosomal elements and an egfp gene. Sf9 insect cells were transfected with AcMNPV-WIV-Syn1 DNA and progeny virus was examined by electron microscopy, and assayed in one-step growth curves and oral infectivity. The results conclusively showed that the rescued virus AcMNPV-WIV-Syn1 had structural and biological properties comparable to the parental virus. We validated a proof of concept that a bona fide baculovirus can be synthesized. The new platform allows manipulation at any or multiple loci and will facilitate future studies such as identifying the minimal baculovirus genome and construction of better expression vectors. This is the largest DNA virus synthesized so far, and its success is likely to be the impetus to stimulate the fields of other large DNA viruses such as herpesviruses and poxviruses.

The Host Specificities of Baculovirus per os Infectivity Factors.

Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host's midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses.

Genome Sequencing and Analysis of Catopsilia pomona nucleopolyhedrovirus: A Distinct Species in Group I Alphabaculovirus.

The genome sequence of Catopsilia pomona nucleopolyhedrovirus (CapoNPV) was determined by the Roche 454 sequencing system. The genome consisted of 128,058 bp and had an overall G+C content of 40%. There were 130 hypothetical open reading frames (ORFs) potentially encoding proteins of more than 50 amino acids and covering 92% of the genome. Among all the hypothetical ORFs, 37 baculovirus core genes, 23 lepidopteran baculovirus conserved genes and 10 genes conserved in Group I alphabaculoviruses were identified. In addition, the genome included regions of 8 typical baculoviral homologous repeat sequences (hrs). Phylogenic analysis showed that CapoNPV was in a distinct branch of clade "a" in Group I alphabaculoviruses. Gene parity plot analysis and overall similarity of ORFs indicated that CapoNPV is more closely related to the Group I alphabaculoviruses than to other baculoviruses. Interesting, CapoNPV lacks the genes encoding the fibroblast growth factor (fgf) and ac30, which are conserved in most lepidopteran and Group I baculoviruses, respectively. Sequence analysis of the F-like protein of CapoNPV showed that some amino acids were inserted into the fusion peptide region and the pre-transmembrane region of the protein. All these unique features imply that CapoNPV represents a member of a new baculovirus species.

Genome-wide analysis of differential mRNA expression of Amsacta moorei entomopoxvirus, mediated by the gene encoding a viral protein kinase (AMV197).

Insect-born entomopoxviruses (Fam: Poxviridae) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus. Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha (ef1a) gene) was downregulated in the absence of amv197. Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process.

Establishment of a cell line from the ash and privet borer beetle Tylonotus bimaculatus Haldeman and assessment of its sensitivity to diacylhydrazine insecticides.

A novel cell line, NRCAN-Tb521, was developed from larvae of the longhorn beetle Tylonotus bimaculatus (Coleoptera: Cerambycidae), a pest of North American ash trees. The cell line has been successfully passaged more than 50 times and displayed very strong attachment to the substrate and a modal chromosomal count distribution of 19. Sequencing of a 649 bp fragment of the mitochondrial cytochrome oxidase I gene confirmed the identity of NRCAN-Tb521 as T. bimaculatus. The response of the cell line to 20-hydroxyecdysone and diacylhydrazine ecdysone agonist insecticides was also studied. At 10(-6) M, 20-hydroxyecdysone, tebufenozide, methoxyfenozide and halofenozide triggered the production of numerous filamentous cytoplasmic extensions, and the cells tended to form aggregates, indicative of a cell differentiation response. This response was followed by a strong decrease in viability after 4 d. Reverse transcription polymerase chain reaction (PCR) experiments and sequencing of PCR fragments showed that the 20E receptor gene EcR is expressed in the cells and that 20E, tebufenozide, methoxyfenozide and halofenozide also induce the expression of the nuclear hormone receptor gene HR3. This report establishes that NRCAN-Tb521 is a valuable in vitro model to study effects of ecdysone agonists in wood-boring cerambycids.

Gene acquisition convergence between entomopoxviruses and baculoviruses.

Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae) and the baculoviruses (BVs). EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host's immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.

Genome sequence and analysis of Buzura suppressaria nucleopolyhedrovirus: a group II Alphabaculovirus.

The genome of Buzura suppressaria nucleopolyhedrovirus (BusuNPV) was sequenced by 454 pyrosequencing technology. The size of the genome is 120,420 bp with 36.8% G+C content. It contains 127 hypothetical open reading frames (ORFs) covering 90.7% of the genome and includes the 37 conserved baculovirus core genes, 84 genes found in other baculoviruses, and 6 unique ORFs. No typical baculoviral homologous repeats (hrs) were present but the genome contained a region of repeated sequences. Gene Parity Plots revealed a 28.8 kb region conserved among the alpha- and beta-baculoviruses. Overall comparisons of BusuNPV to other baculoviruses point to a distinct species in group II Alphabaculovirus.

The ha72 core gene of baculovirus is essential for budded virus production and occlusion-derived virus embedding, and amino acid K22 plays an important role in its function.

ha72 of Helicoverpa armigera nucleopolyhedrovirus (a homologue of ac78) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33.

New insights into the evolution of Entomopoxvirinae from the complete genome sequences of four entomopoxviruses infecting Adoxophyes honmai, Choristoneura biennis, Choristoneura rosaceana, and Mythimna separata.

Poxviruses are nucleocytoplasmic large DNA viruses encompassing two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae, infecting vertebrates and insects, respectively. While chordopoxvirus genomics have been widely studied, only two entomopoxvirus (EPV) genomes have been entirely sequenced. We report the genome sequences of four EPVs of the Betaentomopoxvirus genus infecting the Lepidoptera: Adoxophyes honmai EPV (AHEV), Choristoneura biennis EPV (CBEV), Choristoneura rosaceana EPV (CREV), and Mythimna separata EPV (MySEV). The genomes are 80% AT rich, are 228 to 307 kbp long, and contain 247 to 334 open reading frames (ORFs). Most genes are homologous to those of Amsacta moorei entomopoxvirus and encode several protein families repeated in tandem in terminal regions. Some genomes also encode proteins of unknown functions with similarity to those of other insect viruses. Comparative genomic analyses highlight a high colinearity among the lepidopteran EPV genomes and little gene order conservation with other poxvirus genomes. As with previously sequenced EPVs, the genomes include a relatively conserved central region flanked by inverted terminal repeats. Protein clustering identified 104 core EPV genes. Among betaentomopoxviruses, 148 core genes were found in relatively high synteny, pointing to low genomic diversity. Whole-genome and spheroidin gene phylogenetic analyses showed that the lepidopteran EPVs group closely in a monophyletic lineage, corroborating their affiliation with the Betaentomopoxvirus genus as well as a clear division of the EPVs according to the orders of insect hosts (Lepidoptera, Coleoptera, and Orthoptera). This suggests an ancient coevolution of EPVs with their insect hosts and the need to revise the current EPV taxonomy to separate orthopteran EPVs from the lepidopteran-specific betaentomopoxviruses so as to form a new genus.

Induction of apoptosis by the Amsacta moorei entomopoxvirus.

CF-70-B2 cells derived from the spruce budworm (Choristoneura fumiferana) undergo apoptosis when infected with Amsacta moorei entomopoxvirus (AMEV), as characterized by membrane blebbing, formation of apoptotic bodies, TdT-mediated dUTP nick-end labelling (TUNEL) staining, condensed chromatin and induction of caspase-3/7 activity. The apoptotic response was reduced when cells were infected with UV-inactivated AMEV, but not when infected in the presence of the DNA synthesis inhibitor, cytosine ß-d-arabinofuranoside. Hence, only pre-DNA replication events were involved in inducing the antiviral response in CF-70-B2 cells. The virus eventually overcame the host's antiviral response and replicated to high progeny virus titres accompanied by high levels of caspase-3/7 activity. The CF-70-B2 cells were less productive of progeny virus in comparison to LD-652, a Lymantria dispar cell line routinely used for propagation of AMEV. At late stages of infection, LD-652 cells also showed characteristics of apoptosis such as oligosomal DNA fragmentation, TUNEL staining, condensed chromatin and increased caspase-3/7 activity. Induction of apoptosis in LD-652 cells was dependent on viral DNA replication and/or late gene expression. A significantly reduced rate of infection was observed in the presence of general caspase inhibitors Q-VD-OPH and Z-VAD-FMK, indicating caspases may be involved in productive virus infection.

Role of interactions between Autographa californica multiple nucleopolyhedrovirus procathepsin and chitinase chitin-binding or active-site domains in viral cathepsin processing.

The binding of Autographa californica multiple nucleopolyhedrovirus chitinase (CHIA) to viral cathepsin protease progenitor (proV-CATH) governs cellular/endoplasmic reticulum (ER) coretention of CHIA and proV-CATH, thus coordinating simultaneous cellular release of both host tissue-degrading enzymes upon host cell death. CHIA is a proposed proV-CATH folding chaperone because insertional inactivation of chiA causes production of proV-CATH aggregates that are incompetent for proteolytic maturation into active V-CATH enzyme. We wanted to determine whether the N-terminal chitin-binding domain (CBD, 149 residues) and C-terminal CHIA active-site domain (ASD, 402 residues) of CHIA bind to proV-CATH independently of one another and whether either domain is dispensable for CHIA's putative proV-CATH folding chaperone activity. We demonstrate that N-terminally green fluorescent protein (GFP)-fused CHIA, ASD, and CBD each colocalize with proV-CATH-RFP in ER-like patterns and that both ASD and CBD independently associate with proV-CATH in vivo using bimolecular fluorescence complementation (BiFC) and in vitro using reciprocal nickel-histidine pulldown assays. Altogether, the data from colocalization, BiFC, and reciprocal copurification analyses suggest specific and independent interactions between proV-CATH and both domains of CHIA. These data also demonstrate that either CHIA domain is dispensable for normal proV-CATH processing. Furthermore, in contrast to prior evidence suggesting that a lack of chiA expression causes proV-CATH to become aggregated, insoluble, and unable to mature into V-CATH, a chiA deletion bacmid virus we engineered to express just v-cath produced soluble proV-CATH that was prematurely secreted from cells and proteolytically matured into active V-CATH enzyme.

Cell-dependent production of polyhedra and virion occlusion of Autographa californica multiple nucleopolyhedrovirus fp25k mutants in vitro and in vivo.

Members of the family Baculoviridae are insect-specific dsDNA viruses that have been used for biological control of insect pests in agriculture and forestry, as well as in research and pharmaceutical protein expression in insect cells and larvae. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of the family Baculoviridae. During infection of AcMNPV in permissive cells, fp25k mutants are positively selected, leading to the formation of the few polyhedra (FP) phenotype with reduced yield of polyhedra and reduced virion occlusion efficiency, which leads to decreased oral infectivity for insects. Here we report that polyhedra of AcMNPV fp25k mutants produced from different insect cell lines and insects have differences in larval per os infectivity, and that these variations are due to different virion occlusion efficiencies in these cell lines and insects. Polyhedra of AcMNPV fp25k mutants produced from Sf cells (Sf21 and Sf9, derived from Spodoptera frugiperda) and S. frugiperda larvae had poorer virion occlusion efficiency than those from Hi5 cells (derived from Trichoplusia ni) and T. ni larvae, based on immunoblots, DNA isolation and larval oral infection analysis. AcMNPV fp25k mutants formed clusters of FP and many polyhedra (MP) in the fat body cells of both T. ni and S. frugiperda larvae. Transmission electron microscopy revealed that the nature of virion occlusion of AcMNPV fp25k mutants was dependent on the different cells of the T. ni fat body tissue. Taken together, these results indicate that the FP phenotype and virion occlusion efficiency of fp25k mutants are influenced by the host insect cells.

Insect cell culture: virus replication and applications in biotechnology.

Insect cell lines have been initiated since the 1930s and were used to replicate insect baculoviruses as well as arboviruses. Since the latter group of viruses cause serious diseased in man and equines, efforts were expended to characterize the viruses in the new cell lines in attempts to understand the replication cycle at the cellular and molecular levels. Soon it was realized that insect baculoviruses have a potential as viable alternatives to chemicals in the control of agricultural and forest insect pests. The cell lines provided excellent tools to understand the molecular biology of baculoviruses before wide-scale use in the field. During these investigastions, it came to light that baculoviruses can be exploited as vectors for the expression of exogenous proteins and vaccines. The amenability of the virus to genetic modifications and the increasing numbers of permissive cell lines opened new avenues in protein expression. However, not all baculoviruses were able to replicate in cell lines. Indeed, there are no cell lines permissive to viruses belonging to the genera Gammabaculvirus and Deltabaculovirus. Some entomopoxviruses have been replicated in a few cell lines and this paper reports the replication of an entomopoxvirus from the spruce budworm in a homologous cell line.