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In December 1983 a seminal paper appeared on the overexpression of human interferon-ß in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produce massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes are dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years major improvements have been achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells, and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted baculovirus expression vectors are used to deliver and express heterologous genes in mammalian cells and may find applications for gene therapy and cancer treatment in the future. The purpose of this paper is to highlight the 30-years 'anniversary' of this expression system by summarizing the fundamental research that allowed the development of this expression system and by indicating the major technological advances since 1983. Finally, attention will be paid to the future challenges to further optimize this amazing technology.

Faster-acting recombinant baculoviruses have shown potential for improved suppression of insect pests, but their ecological impact on target and nontarget hosts and naturally occurring pathogens needs to be assessed. Previous studies have focused on the fitness of recombinants at the between-hosts level. However, the population structure of the transmission stages will also be decided by within-host selection. Here we have experimentally quantified the within-host competitive fitness of a fast-acting recombinant Autographa californica multicapsid nucleopolyhedrovirus missing the endogenous egt gene (vEGTDEL), by means of direct competition in single- and serial-passage experiments with its parental virus. Quantitative real-time PCR was employed to determine the ratio of these two viruses in passaged mixtures. We found that vEGTDEL had reduced within-host fitness: per passage the ratio of wild type to vEGTDEL was on average enhanced by a factor of 1.53 (single passage) and 1.68 (serial passage). There is also frequency-dependence: the higher the frequency of vEGTDEL, the stronger the selection against it is. Additionally, the virus ratio is a predictor of time to host death and virus yield. Our results show that egt is important to within-host fitness and allow for a more complete assessment of the ecological impact of recombinant baculovirus release.

The Autographa californica nucleopolyhedrovirus (AcMNPV) contains three apoptosis suppressor genes: p35, iap1 and iap2. AcMNPV P35 functions as a pancaspase inhibitor, but the function of IAP1 and IAP2 has not been entirely resolved. In this paper, we analyze the function of IAP1 and IAP2 in detail. AcMNPV with p35-deletion inhibited the apoptosis of BTI-Tn-5B1-4 (Tn-Hi5) cells induced by a Helicoverpa armigera single nucleocapsid NPV (HearNPV) infection and rescued the replication of HearNPV and BV production in these cells. Transient-expression experiments indicated that both IAP1 and IAP2 suppress apoptosis of Tn-Hi5 cells during HearNPV infection. Recombinant HearNPVs expressing AcMNPV iap1, iap2 and p35, respectively, not only prevented apoptosis but also allowed HearNPV to replicate in Tn-Hi5 cells. However, the iap1, iap2 and p35 genes when expressed in HearNPV were unable to rescue BV production. These results indicate that both AcMNPV iap1 and iap2 function independently as apoptosis inhibitors of and are potential host range factors.

The genus Nucleopolyhedrovirus (NPV) of the family Baculoviridae can be subdivided phylogenetically into two groups. The same division can be made on the basis of their budded virus (BV) envelope fusion protein. Group I NPVs are characterized by the presence of a GP64-like major envelope fusion protein, which is involved in viral attachment and the fusion of virus and cell membrane, and is required for budding of progeny nucleocapsids. Group II NPVs have an envelope fusion protein unrelated to GP64, named F. In contrast to GP64, F proteins are found in all baculoviruses, but they are not functional as envelope fusion proteins in group I NPVs. Autographa californica multiple NPV (AcMNPV) lacking GP64 can be pseudotyped by the F protein of Spodoptera exigua multiple NPV (SeMNPV), suggesting that F proteins are functionally analogous to GP64. GP64 homologues are thought to have been acquired by group I NPVs during evolution, thereby giving these viruses a selective advantage and obviating the need for a functional F protein. To address this supposition experimentally, attempts were made to pseudotype a group II NPV, SeMNPV, with GP64. Transfection of an f-null SeMNPV bacmid into Se301 cells did not result in the production of infectious BVs. This defect was rescued by insertion of SeMNPV f, but not by insertion of AcMNPV gp64. This suggests that the functional analogy between GP64 and F is not readily reciprocal and that F proteins from group II NPVs may provide additional functions in BV formation that are lacking in the GP64 type of fusion protein.

F proteins of group II nucleopolyhedroviruses (NPVs) are envelope fusion proteins essential for virus entry and egress. An F-null Helicoverpa armigera single nucleocapsid NPV (HearNPV) bacmid, HaBacF, was constructed. This bacmid could not produce infectious budded virus (BV) when transfected into HzAM1 cells, showing that F protein is essential for cell-to-cell transmission of BVs. When HaBacF was pseudotyped with the homologous F protein (HaBacF-HaF, positive control) or with the heterologous F protein from Spodoptera exigua multinucleocapsid NPV (SeMNPV) (HaBacF-SeF), infectious BVs were produced with similar kinetics. In the late phase of infection, the BV titre of HaBacF-SeF virus was about ten times lower than that of HaBacF-HaF virus. Both pseudotyped viruses were able to fuse HzAM1 cells in a similar fashion. The F proteins of both HearNPV and SeMNPV were completely cleaved into F1 and F2 in the BVs of vHaBacF-HaF and vHaBacF-SeF, respectively, but the cleavage of SeF in vHaBacF-SeF-infected HzAM1 cells was incomplete, explaining the lower BV titre of vHaBacF-SeF. Polyclonal antisera against HaF1 and SeF1 specifically neutralized the infection of vHaBacF-HaF and vHaBacF-SeF, respectively. HaF1 antiserum showed some cross-neutralization with vHaBacF-SeF. These results demonstrate that group II NPV F proteins can be functionally replaced with a homologue of other group II NPVs, suggesting that the interaction of F with other viral or host proteins is not absolutely species-specific.

Oryctes rhinoceros virus (OrV) is an unassigned invertebrate dsDNA virus with enveloped and rod-shaped virions. Two cloned PstI fragments, C and D, of OrV DNA have been sequenced, consisting of 19,805 and 17,146¿bp, respectively, and comprising about 30% of the OrV genome. For each of the two fragments, 20 open reading frames (ORFs) of 150 nucleotides or greater with no or minimal overlap were predicted. Ten of the predicted 40 ORFs revealed significant similarities to Heliothis zea virus 1 (HzV-1) ORFs, of which five, lef-4, lef-5, pif-2, dnapol and ac81, are homologues of conserved core genes in the family Baculoviridae, and one is homologous to baculovirus rr1. A baculovirus odv-e66 homologue is also present in OrV. Five ORFs encode proteins homologous to cellular thymidylate synthase (TS), patatin-like phospholipase, mitochondrial carrier protein, Ser/Thr protein phosphatase, and serine protease, respectively. TS is phylogenetically related to those of eukarya and nucleo-cytoplasmic large dsDNA viruses. However, the remaining 25 ORFs have poor or no sequence matches with the current databases. Both the gene content of the sequenced fragments and the phylogenetic analyses of the viral DNA polymerase suggest that OrV is most closely related to HzV-1. These findings and the re-evaluation of the relationship of HzV-1 to baculoviruses suggest that a new virus genus, Nudivirus, should be established, containing OrV and HzV-1, which are genetically related to members of the family Baculoviridae.

Contains fulltext : 52851.pdf (Publisher’s version ) (Closed access) Insect larvae spend most of their time eating and the digestive tract is the most crucial barrier for the entrance of many pathogens. In our study, suppression subtractive hybridization (SSH) was used to compare Spodoptera exigua midgut gene expression between larvae exposed to the Bacillus thuringiensis Cry1Ca toxin and non-exposed insects. Based on the SSH results, full cDNA sequences coding for four homologous proteins were obtained. Quantitative and semi-quantitative RT-PCR showed the increased expression of the genes coding for these proteins after exposure to different B. thuringiensis toxins as well as after infection with baculovirus. The proteins were named REPAT after their increased expression in Response to Pathogen. REPAT1, a member of this family, was recombinantly expressed using the baculovirus expression system, revealing the glycosylated nature of the protein. Recombinant baculoviruses expressing REPAT1 were used to infect larvae from S. exigua, showing that expression of REPAT1 was reducing the virulence of baculovirus to the infected larvae. Together, these results suggest a role for REPAT1 in mitigating pathological effects.

Baculovirus genomes are covalently closed circles of double stranded-DNA varying in size between 80 and 180 kilobase-pair. The genomes of more than fourty-one baculoviruses have been sequenced to date. The majority of these (37) are pathogenic to lepidopteran hosts; three infect sawflies (Hymenoptera) and one has a mosquito host (Diptera). With this information, general patterns of genome structure and gene content became apparent. Baculovirus open reading frames are tightly packed with minimal intergenic regions and the coding sequences are almost equally distributed over both strands. Baculovirus genes form single transcription units, with early and late transcribed ORFs scattered along the genome. A set of twenty nine core genes is conserved and therefore is characteristic for baculoviruses. Most baculovirus genomes contain multiple homologous regions with repeated sequences and often palindromic motifs, which play a crucial role as enhancers of early transcription and most likely in viral DNA replication. Baculovirus genomes have a certain degree of plasticity, as evidenced from the genomic variations within virus isolates from the field. Recombination events and transposon insertions appear to play a role in the uptake of new genes from co-infecting viruses or from the insect host. This review deals with the structural and functional properties of baculovirus genomes including both conserved and variable genes.

Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.

The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.

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, viruslike 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.

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).

The turnip moth, Agrotis segetum (Lepidoptera, Noctuidae), is an important pest insect in Europe, Asia, and Africa. We have genetically characterized and classified a nucleopolyhedrovirus isolated from A. segetum larvae in Poland (AgseNPV-P). The restriction pattern of AgseNPV-P was distinct from an isolate from England/France (AgseNPV-UK and AgseNPV-F). Sequence analysis of three conserved baculovirus genes, polyhedrin, lef-8 and pif-2, revealed that AgseNPV-P differs substantially from the already described NPVs isolated from A. segetum and possibly represents a new NPV species. Phylogenetic analysis placed AgseNPV-P among group II NPVs and showed the closest relationship to Agrotis ipsilon (Agip) NPV and Spodoptera exigua (Se) MNPV.

White spot syndrome virus (WSSV), member of a. new virus family called Nimaviridae, is a major scourge in worldwide shrimp, cultivation. Geographical isolates of WSSV identified so far are very similar in morphology and proteome, and show little difference in restriction fragment length polymorphism (RFLP) pattern. We have mapped the genomic differences between three completely sequenced WSSV isolates, originating from Thailand (WSSV-TH), China (WSSV-CN) and Taiwan (WSSV-TW). Alignment of the genomic sequences of these geographical isolates revealed an overall nucleotide identity of 99.32%. The major difference among the three isolates is a deletion of approximately 13 kb (WSSV-TH) and 1 kb (WSSV-CN), present in the same genomic region, relative to WSSV-TW. A second difference involves a genetically variable region of about 750 bp. All other variations >2 bp between the three isolates are located in repeat regions along the genome. Except for the homologous regions (hrl, hr3, hr8 and hr9), these variable repeat regions are almost exclusively located in ORFs, of which the genomic repeat regions in ORF75, ORF94 and ORF125 can be used for PCR based classification of WSSV isolates in epidemiological studies. Furthermore, the comparison identified highly invariable genomic loci, which may be used for reliable monitoring of WSSV infections and for shrimp health certification.

Open reading frame 94 (Ha94) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) is 1086 bp long and a homologue of Autographa californica multiple NPV ORF109. The gene is conserved among all baculoviruses whose genomes have been completely sequenced so far and is thus considered a baculovirus core gene. Ha94 transcripts were detected from 24 to 96 h post-infection (p.i.) of HzAM1 cells with HaSNPV. Polyclonal antiserum raised to a GST-HA94 fusion protein recognized a 43 kDa protein, HA94, in infected cell lysates from 36 to 96 h p.i., suggesting that Ha94 is a late gene. Western blot analysis of proteins present in budded virus and occlusion-derived virus (ODV) showed that Ha94 encodes a structural component of ODV. When ODVs were fractionated further into nucleocapsid and envelope components, Western blot analysis indicated that the encoded protein was associated with both the nucleocapsid and the envelope. In summary, data available indicated that Ha94 encodes a novel ODV-specific protein of HaSNPV, designated ODV-EC43.

Complete cDNA sequences were obtained for ribosomal protein (rp) L15 and eukaryotic initiation factor eIF2alpha from the lepidopteran insect Spodoptera frugiperda, and for elongation factor eEF2 from S. exigua. The presence of a 5' terminal oligopyrimidine (TOP) tract classified the lepidopteran rpL15 transcript as a TOP mRNA. For eEF2, two types of transcripts were observed, one of which had a 5'TOP tract. The transcript levels for rpL15, eEF2 and eIF2alpha decreased following baculovirus infection. Polysome analysis showed that the corresponding mRNAs remained to be translated until at least 16 h post-infection for both TOP and non-TOP mRNAs. Baculovirus-induced host shut-off therefore appears to be regulated at the level of RNA abundance rather than at the translational level.

Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from insect hosts have been previously identified. In order to gain insight into DNA replication of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV, Groups II), the essential cis-acting DNA segments were studied. A strain, named Se-4, was plaque-purified from SeMNPV isolate US1. PCR, ELT-PCR and REN showed that Se-4 was genetically relatively homogeneous and retained the full-length of a hypervariable region which is usually prone to deletion from SeMNPV genome. To study the stability of this isolate in vitro, Se-4 was serially passaged in the Se301 cell line up to 10 times without dilution. Intracellular viral DNA extracted from every passage was analyzed by REN. A novel 3.5 kb PstI fragment was observed in passage 7 and the relative intensities of the bands increased with subsequent passages. In passage 10, the molar ratio of the fragment was much higher than those of any other viral DNA fragments. This fragment was thus expected to contain an important cis-acting element for SeMNPV DNA replication. The fragment was cloned and sequenced and it was found that it overlapped 3 525 bp with the published SeMNPV genome sequence (GenBank AF169823), from 81 014 nt to 84 538 nt. The region contained the SeMNPV non-hr origin (ori) of DNA replication and some ORFs including partial vlf-1, partial p26, Se84 as well asSe83, Se85, Se86, which are unique to SeMNPV. As compared to Autographa californica MNPV (Groups I), which also generated hypermolar DNA fragments containing the non-hr ori by serial undiluted passage in IPLB-SF-21 cell culture, our results provided in vitro evidence that the non-hr ori may also play an important role in the viral infection cycle of Groups II NPVs

Key virus traits such as virulence and transmission strategies rely on genetic variation that results in functional changes in the interactions between hosts and viruses. Here, comparative genomic analyses of seven isolates of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) with differing phenotypes were employed to pinpoint candidate genes that may be involved in host– virus interactions. These isolates obtained after vertical or horizontal transmission of infection in insects differed in virulence. Apart from one genome containing a piggyBac transposon, all European SeMNPV isolates had a similar genome size and content. Complete genome analyses of single nucleotide polymorphisms and insertions/deletions identified mutations in 48 ORFs that could result in functional changes. Among these, 13 ORFs could be correlated with particular phenotypic characteristics of SeMNPV isolates. Mutations were found in all gene functional classes and most of the changes we highlighted could potentially be associated with differences in transmission. The regulation of DNA replication (helicase, lef-7) and transcription (lef-9, p47) might be important for the establishment of sublethal infection prior to and following vertical transmission. Virus–host cell interactions also appear instrumental in the modulation of viral transmission as significant mutations were detected in virion proteins involved in primary (AC150) or secondary infections (ME35) and in apoptosis inhibition (IAP2, AC134). Baculovirus populations naturally harbour high genomic variation located in genes involved at different levels of the complex interactions between virus and host during the course of an infection. The comparative analyses performed here suggest that the differences in baculovirus virulence and transmission phenotypes involve multiple molecular pathways., This study received financial support from the Spanish Ministry for Science and Technology (AGL2005-07909-CO3-01, AGL2008-05456- C03-01). O. C. received a Predoctoral Fellowship from the Spanish Ministry of Education and Culture. J. T. and E. A. H. are supported by the European Research Council (grant 205206 GENOVIR).

The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and in their egress from the virogenic stroma toward the nuclear periphery by a mechanism, which also includes F-actin filaments. We performed functional mapping of VP80 demonstrating that its highly conserved C-terminal region plays a crucial role in virion morphogenesis. Protein database mining identified a putative basic helix-loop-helix (bHLH) domain, a DNA-binding module typical for eukaryotic transcription factors, in the essential C-terminal region of VP80. Using a molecular modeling approach, we predicted the three-dimensional structure of this domain, revealing some unique properties. Biochemical assays proved that VP80 can form homodimers, a critical prerequisite of DNA-binding bHLH proteins. The ability of VP80 to bind DNA was subsequently confirmed by an electrophoretic mobility shift assay. We further show that AcMNPV DNA replication occurs in the absence of VP80. Immunolabeling of VP80 in baculovirus-infected cells rather points toward its involvement in nucleocapsid maturation. The competence of VP80 to interact with both F-actin and DNA provides novel insight into baculovirus morphogenesis.

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.

In this review background information on the baculovirus-insect cell expression system and its applications for producing protein subunits and virus-like particles for vaccine and other purposes is provided. This review will illustrate the principle structure of baculovirus vectors commonly used for heterologous gene expression in insect cells and describe adaptations that have been made over the last 10 years to improve the system in terms of quality of the protein produced and stability of the baculovirus genome. These improvements include enhanced trafficking, folding and glycosylation of the recombinant protein as well as preventing intracellular degradation. Challenges and progress in stabilizing the baculovirus genome in order not to lose the transgene cassette will also be discussed. Recent developments such as how to make multiple alterations in the baculovirus genome without accumulating marker genes are included.

The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9 -null mutant of Autographa californica multiple nucleopolyhedrovirus ( Ac MNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alpha baculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus , comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that Ac MNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9 -null Ac MNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly.

A new cell line was established from the embryos of the insect Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). The cell line contains several morphologically different cell types and was distinguished from three other lepidopteran cell lines propagated in the laboratory by DNA amplification fingerprinting. The cultured cells, which we officially named WU-CcE-1 cells, were permissive for infection by C. chalcites nucleopolyhedrovirus (ChchNPV) and large numbers of occlusion bodies were produced that retained their infectivity for C. chalcites larvae. The CcE-1 cells were also permissive for Trichoplusia ni single nucleopolyhedrovirus (TnSNPV). ChchNPV could be passaged in these cells for at least four passages indicating that budded virus production was supported. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa armigera (Hear) NPV both induced apoptosis in these cells. The results obtained indicate that the CcE-1 cell line will be a useful tool in the study of both ChchNPV and TnSNPV.

The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome-independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin-specific inhibitor 2,3-butanedione monoxime (BDM) and the microtubule inhibitor nocodazole, the low pH-triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. The reverse transcription-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH-mediated infection assay and VP39 and EEA1 colocalization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low-pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64 -null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64 -rescued group II NPV of Helicoverpa armigera (vHaBacΔF-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBacΔF-gp64 compared to the HaF rescue control virus vHaBacΔF-HaF. Electron microscopy further showed that most vHaBacΔF-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBacΔF-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.

Faster-acting recombinant baculoviruses have shown potential for improved suppression of insect pests, but their ecological impact on target and nontarget hosts and naturally occurring pathogens needs to be assessed. Previous studies have focused on the fitness of recombinants at the between-hosts level. However, the population structure of the transmission stages will also be decided by within-host selection. Here we have experimentally quantified the within-host competitive fitness of a fast-acting recombinant Autographa californica multicapsid nucleopolyhedrovirus missing the endogenous egt gene (vEGTDEL), by means of direct competition in single- and serial-passage experiments with its parental virus. Quantitative real-time PCR was employed to determine the ratio of these two viruses in passaged mixtures. We found that vEGTDEL had reduced within-host fitness: per passage the ratio of wild type to vEGTDEL was on average enhanced by a factor of 1.53 (single passage) and 1.68 (serial passage). There is also frequency-dependence: the higher the frequency of vEGTDEL, the stronger the selection against it is. Additionally, the virus ratio is a predictor of time to host death and virus yield. Our results show that egt is important to within-host fitness and allow for a more complete assessment of the ecological impact of recombinant baculovirus release.

Several species of tsetse flies can be infected by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). Infection causes salivary gland hypertrophy and also significantly reduces the fecundity of the infected flies. To better understand the molecular basis underlying the pathogenesis of this unusual virus, we sequenced and analyzed its genome. The GpSGHV genome is a double-stranded circular DNA molecule of 190,032 bp containing 160 nonoverlapping open reading frames (ORFs), which are distributed equally on both strands with a gene density of one per 1.2 kb. It has a high A+T content of 72%. About 3% of the GpSGHV genome is composed of 15 sequence repeats, distributed throughout the genome. Although sharing the same morphological features (enveloped rod-shaped nucleocapsid) as baculoviruses, nudiviruses, and nimaviruses, analysis of its genome revealed that GpSGHV differs significantly from these viruses at the level of its genes. Sequence comparisons indicated that only 23% of GpSGHV genes displayed moderate homologies to genes from other invertebrate viruses, principally baculoviruses and entomopoxviruses. Most strikingly, the GpSGHV genome encodes homologues to the four baculoviral per os infectivity factors (p74 [pif-0], pif-1, pif-2, and pif-3). The DNA polymerase encoded by GpSGHV is of type B and appears to be phylogenetically distant from all DNA polymerases encoded by large double-stranded DNA viruses. The majority of the remaining ORFs could not be assigned by sequence comparison. Furthermore, no homologues to DNA-dependent RNA polymerase subunits were detected. Taken together, these data indicate that GpSGHV is the prototype member of a novel group of insect viruses.

F proteins of group II nucleopolyhedroviruses (NPVs) are envelope fusion proteins essential for virus entry and egress. An F-null Helicoverpa armigera single nucleocapsid NPV (HearNPV) bacmid, HaBacF, was constructed. This bacmid could not produce infectious budded virus (BV) when transfected into HzAM1 cells, showing that F protein is essential for cell-to-cell transmission of BVs. When HaBacF was pseudotyped with the homologous F protein (HaBacF-HaF, positive control) or with the heterologous F protein from Spodoptera exigua multinucleocapsid NPV (SeMNPV) (HaBacF-SeF), infectious BVs were produced with similar kinetics. In the late phase of infection, the BV titre of HaBacF-SeF virus was about ten times lower than that of HaBacF-HaF virus. Both pseudotyped viruses were able to fuse HzAM1 cells in a similar fashion. The F proteins of both HearNPV and SeMNPV were completely cleaved into F1 and F2 in the BVs of vHaBacF-HaF and vHaBacF-SeF, respectively, but the cleavage of SeF in vHaBacF-SeF-infected HzAM1 cells was incomplete, explaining the lower BV titre of vHaBacF-SeF. Polyclonal antisera against HaF1 and SeF1 specifically neutralized the infection of vHaBacF-HaF and vHaBacF-SeF, respectively. HaF1 antiserum showed some cross-neutralization with vHaBacF-SeF. These results demonstrate that group II NPV F proteins can be functionally replaced with a homologue of other group II NPVs, suggesting that the interaction of F with other viral or host proteins is not absolutely species-specific.

Oryctes rhinoceros virus (OrV) is an unassigned invertebrate dsDNA virus with enveloped and rod-shaped virions. Two cloned PstI fragments, C and D, of OrV DNA have been sequenced, consisting of 19,805 and 17,146¿bp, respectively, and comprising about 30% of the OrV genome. For each of the two fragments, 20 open reading frames (ORFs) of 150 nucleotides or greater with no or minimal overlap were predicted. Ten of the predicted 40 ORFs revealed significant similarities to Heliothis zea virus 1 (HzV-1) ORFs, of which five, lef-4, lef-5, pif-2, dnapol and ac81, are homologues of conserved core genes in the family Baculoviridae, and one is homologous to baculovirus rr1. A baculovirus odv-e66 homologue is also present in OrV. Five ORFs encode proteins homologous to cellular thymidylate synthase (TS), patatin-like phospholipase, mitochondrial carrier protein, Ser/Thr protein phosphatase, and serine protease, respectively. TS is phylogenetically related to those of eukarya and nucleo-cytoplasmic large dsDNA viruses. However, the remaining 25 ORFs have poor or no sequence matches with the current databases. Both the gene content of the sequenced fragments and the phylogenetic analyses of the viral DNA polymerase suggest that OrV is most closely related to HzV-1. These findings and the re-evaluation of the relationship of HzV-1 to baculoviruses suggest that a new virus genus, Nudivirus, should be established, containing OrV and HzV-1, which are genetically related to members of the family Baculoviridae.

Background White Spot Syndrome Virus, a member of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustacean species. Although limited information is available on the mode of transcription, previous data suggest that WSSV gene expression occurs in a coordinated and cascaded fashion. To search in silico for conserved promoter motifs (i) the abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined, and (ii) a MEME search was performed in the upstream sequences of either early or late WSSV genes, as assigned by microarray analysis. Both methods were validated by alignments of empirically determined 5' ends of various WSSV mRNAs. Results The collective information shows that the upstream region of early WSSV genes, containing a TATA box and an initiator, is similar to Drosophila RNA polymerase II core promoter sequences, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known well-established late genes, including all major structural protein genes, identified a degenerate motif (ATNAC) which could be involved in WSSV late transcription. For these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as previously assigned by microarray analysis, did contain a TATA box in their upstream region. Conclusion The data may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism.

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, viruslike 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.

A recombinant baculovirus (HaSNPV-AaIT) with improved insecticidal properties was constructed for the control of the cotton bollworm (Helicoverpa armigera). A chimeric promoter of the p6.9 and polyhedrin gene of H. armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) was used to drive the expression of an insect-selective scorpion toxin (AaIT) at the egt gene locus of HaSNPV. This chimeric promoter, denoted ph-p69p, was constructed by directional insertion of the p6.9 promoter downstream of the polyhedrin promoter. Laboratory bioassays indicate that the infectivity (LD(50)s) of this recombinant is unchanged, compared to one wild-type clone of HaSNPV (HaSNPV-WT), and an egt-deletion mutant (HaSNPV-EGTD). The median survival times (ST(50)s) of 1st to 5th instar H. armigera larvae were reduced 17-34% after infection with HaSNPV-AaIT in comparison to HaSNPV-WT. The median times of feeding cessation (FT(50)s) Were 30-43% shorter for HaSNPV-AaIT than for HaSNPV-WT in the 3rd to 5th instar of this species. This virus acts also quicker than HaSNPV-EGTD. Field trials at two research sites in 2000 indicate that the number of larvae and the proportion of damaged squares, flowers, and bolls was significantly lower in cotton plots treated with HaSNPV-AaIT than in plots treated with HaSNPV-WT or HaSNPV-EGTD. When HaSNPV-AaIT was applied to control infestations of bollworm over an entire cotton season, yield of cotton lint in plots treated by this recombinant was 22.1% higher than that in HaSNPV-WT treated plots in 2001 (p

Summary 1Parasites and pathogens manipulate their hosts in a variety of ways that are thought to enhance their fitness. However, it is rare to be able to link such phenotypic changes to specific genes. 2Here the effect of a single pathogen gene is examined. The ecdysteroid UDP-glucosyltransferase (egt) gene of insect baculoviruses produces an enzyme that interferes with host moulting. 3The effect of the egt gene was examined by comparing two baculoviruses that differed only in the expression of this gene. All three fitness traits examined – pathogenicity, infection duration and pathogen productivity – were affected by deletion of the gene. 4Trichoplusia ni larvae in all five instars died earlier when infected with the egt-minus virus compared with those infected by the wild-type Autographa californica nucleopolyhedrovirus. 5Unexpectedly, the egt-minus virus was more pathogenic to final instar larvae than the wild-type virus. Virus genotype and dose both influenced insect development. 6Wild-type infected insects had a significantly higher yield of virus at death, cadaver weight and yield of virus per unit weight than those infected with the egt-minus virus. 7The size of the virus challenge had a major influence on the outcome of the interaction. The consequences of these data for pathogen fitness are discussed.

Theileria parva is an intracellular protozoan parasite that causes East Coast fever, a severe lymphoproliferative disease in cattle. Previous attempts to produce recombinant sporozoite surface antigen (p67) in bacterial or insect cells for vaccine purposes have not resulted in a correctly folded protein. Here, we report the expression of N- and C-terminal domains of p67 fused to the baculovirus envelope glycoprotein GP64 by cloning the appropriate p67 cDNA segments between the signal sequence and the major portion of GP64. To further advance the generation of such recombinants, existing surface display techniques were combined with bacmid technology. Chimeric proteins were present on the surface of budded viruses as judged by immunogold labelling and were exposed on the surface of insect cells, as concluded from immunofluorescence studies of infected, non-fixed insect cells. In non-denaturing dot blot experiments, a strong reaction was obtained between monoclonal TpM12 and baculovirus particles displaying the p67N-GP64 chimeric protein. This antibody, raised against native p67, also specifically recognized the surface of recombinant-infected cells. Apparently, a more native conformation was achieved than when p67 was expressed in E.coli or in conventional baculovirus expression systems. The baculovirus surface expression system, therefore, provides an improved way of expressing this T.parva sporozoite surface protein.

Successive Viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did not show the accumulation of such concatemers. When introduced into SeUCR1 and ScIZD2109 cells, the non-hr ori concatemers generated in Se301 cells were maintained but did not increase. This suggests that the non-hr ori confers a strong selective advantage in Se301 cells, but not or to a lesser extent in the other cell lines. The cell line-specific accumulation of non-hr ori concatemers might be due to a higher intrinsic recombination frequency in Se301 cells and may reflect tissue related differences involving some host cell factor(s). Since non-hr ori concatemers in Se301 cells were more abundant in intracellular than in extracellular viral DNA preparations, episomal replication and the requirement of a minimal DNA size for packaging into micleocapsids is hypothesized. (C) 2003 Elsevier Inc. All rights reserved.

Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression. In particular, Autographa californica multicapsid nucleopolyhedovirus (AcMNPV) DIs are enriched in an origin of viral DNA replication (ori) not associated with the homologous regions (hrs). This non-hr ori is located within the coding sequence of the non-essential p94 gene. We investigated the effect of a deletion of the AcMNPV non-hr ori on the heterologous protein expression levels following serial passage in Sf21 insect cells. Using homologous ET recombination in E. coli, deletions within the p94 gene were made in a bacterial artificial chromosome (BAC) containing the entire AcMNPV genome (bacmid). All bacmids were equipped with an expression cassette containing the green fluorescent protein gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). For the parental (intact) bacmid only, a strong accumulation of DIs with reiterated non-hr oris was observed. This was not observed for the mutants, indicating that removal of the non-hr ori enhanced the genetic stability of the viral genome upon passaging. However, for all passaged viruses it was found that the entire BAC vector including the expression cassette was spontaneously deleted from the viral genome, leading to a rapid decrease in GFP and CSFV-E2 production. The rationale for the (intrinsic) genetic instability of the BAC vector in insect cells and the implications with respect to large-scale production of proteins with bacmid-derived baculoviruses are discussed.

Infection of cultured insect cells with Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) resulted in the generation of mutants with major genomic deletions. Some of the mutants lacked the ability to infect S. exigua larvae per os. The gene(s) responsible for this phenotype in SeMNPV was mapped within a contiguous sequence encoding ORFs 29-35. In this paper we have shown that SeMNPV ORFs 15-35 (including genes encoding cathepsin, chitinase, GP37, PTPT-2, EGT, PKIP-1 and ARIF-1) are not essential for virus replication in cell culture or by in vivo intrahaemocoelic injection. By site-specific deletion mutagenesis of a full-length infectious clone of SeMNPV (bacmid) using ET recombination in E. coli, a series of SeMNPV bacmid mutants with increasing deletions in ORFs 15-35 was generated. Analyses of these mutants indicated that a deletion of SeMNPV ORF35 (Se35) resulted in loss of oral infectivity of polyhedral occlusion bodies. Reinsertion of ORF35 in SeMNPV bacmids lacking Se35 rescued oral infectivity. We propose the name pif-2 for Se35 and its baculovirus homologues (e.g. Autographa californica MNPV ORF22), by analogy to a different gene recently characterized in Spodoptera littoralis NPV, which was designated per os infectivity factor (pif). Similar to the p74 gene, which encodes an essential structural protein of the occlusion-derived virus envelope, pif and pif-2 belong to a group of 30 genes that are conserved among the Baculoviradae.

GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar M NPV (Ld M NPV) and Spodoptera exigua M NPV (Se M NPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an Ac M NPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64 -null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from Ld M NPV or Se M NPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The Ac M NPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.

Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from insect hosts have been previously identified. In order to gain insight into DNA replication of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV, Groups II), the essential cis-acting DNA segments were studied. A strain, named Se-4, was plaque-purified from SeMNPV isolate US1. PCR, ELT-PCR and REN showed that Se-4 was genetically relatively homogeneous and retained the full-length of a hypervariable region which is usually prone to deletion from SeMNPV genome. To study the stability of this isolate in vitro, Se-4 was serially passaged in the Se301 cell line up to 10 times without dilution. Intracellular viral DNA extracted from every passage was analyzed by REN. A novel 3.5 kb PstI fragment was observed in passage 7 and the relative intensities of the bands increased with subsequent passages. In passage 10, the molar ratio of the fragment was much higher than those of any other viral DNA fragments. This fragment was thus expected to contain an important cis-acting element for SeMNPV DNA replication. The fragment was cloned and sequenced and it was found that it overlapped 3 525 bp with the published SeMNPV genome sequence (GenBank AF169823), from 81 014 nt to 84 538 nt. The region contained the SeMNPV non-hr origin (ori) of DNA replication and some ORFs including partial vlf-1, partial p26, Se84 as well asSe83, Se85, Se86, which are unique to SeMNPV. As compared to Autographa californica MNPV (Groups I), which also generated hypermolar DNA fragments containing the non-hr ori by serial undiluted passage in IPLB-SF-21 cell culture, our results provided in vitro evidence that the non-hr ori may also play an important role in the viral infection cycle of Groups II NPVs