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Mutation rates are of key importance for understanding evolutionary processes and predicting their outcomes. Empirical estimates of mutation rate are available for a number of RNA viruses, but few are available for DNA viruses, which tend to have larger genomes. Whilst some viruses have very high mutation rates, lower mutation rates are expected for viruses with large genomes to ensure genome integrity. Alphabaculoviruses are insect viruses with large genomes and often have high levels of polymorphism, suggesting high mutation rates despite evidence of proofreading activity by the replication machinery. Here, we report an empirical estimate of the mutation rate per base per strand copying (s/n/r) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To avoid biases due to selection, we analyzed mutations that occurred in a stable, non-functional genomic insert after five serial passages in Spodoptera exigua larvae. Population bottlenecks, viral mode of replication and thresholds for mutation detection likely affect mutation rate estimates, and we therefore used population genetic models that account for these processes to infer the mutation rate. We estimated a mutation rate of 1×10−7 s/n/r. This estimate was not sensitive to different model assumptions or including whole genome data. The rates at which different classes of mutations accumulate provide good evidence for neutrality of mutations occurring within the inserted region. We therefore present a robust approach for mutation rate estimation for viruses with stable genomes, and strong evidence of a much lower alphabaculovirus mutation rate than supposed based on the high levels of polymorphism observed.Author SummaryVirus populations can evolve rapidly, driven by the large number of mutations that occur during virus replication. It is challenging to measure mutation rates because selection will affect which mutations are observed: beneficial mutations are overrepresented in virus populations, while deleterious mutations are selected against and therefore underrepresented. Few mutation rates have been estimated for viruses with large DNA genomes, and there are no estimates for any insect virus. Here, we estimate the mutation rate for an alphabaculovirus, a virus that infects caterpillars and has a large, 134 kilobase pair DNA genome. To ensure that selection did not bias our estimate of mutation rate, we studied which mutations occurred in a large artificial region inserted into the virus genome, where mutations did not affect viral fitness. We deep sequenced evolved virus populations, and compared the distribution of observed mutants to predictions from a simulation model to estimate mutation rate. We found evidence for a relatively low mutation rate, of one mutation in every 10 million bases replicated. This estimate is in line with expectations for a virus with self-correcting replication machinery and a large genome.

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host’s brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cell culture. An f homologue gene is absent in gammabaculoviruses. Here we characterized the putative F-homologue (Cuni-F), encoded by (ORF) 104 of Culex nigripalpus nucleopolyhedrovirus (CuniNPV), the only deltabaculovirus member. When expressed alone, this protein seems to locate on the cell surface and is able to induce cell-cell fusion. When expressed by an alphabaculovirus (Autographa california nucleopolyhedrovirus), it was found to be incorporated into BVs. Western blot analyses detected the uncleaved Cuni-F0 and the furin-cleaved F1 forms. Treatment of infected cells with tunicamycin showed that Cuni-F contains N-glycans. Mutagenesis analysis identified the canonical furin cleavage site 126RARR129 as being responsible for the cleavage of Cuni-F in insect cells. The collective evidence suggests that CuniNPV encodes a functional F protein.

Baculoviruses, although they infect insects in nature, can transduce a wide variety of mammalian cells and are therefore promising gene therapy vectors. However, baculovirus transduction into many mammalian cells is very inefficient, and the limiting stages and factors remain unknown. An important finding is that a short-duration trigger with low pH can significantly enhance virus transduction efficiency, but the mechanism is poorly understood. Herein, we performed a detailed comparative study on entry mechanisms of the prototypical baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) into insect and mammalian cells. The results showed that AcMNPV could be internalized into mammalian cells efficiently, but fusion in early endosomes (EEs) appeared to be the major obstacle. Measurement of endosomal pH suggested that virus fusion might be restricted under relatively high-pH conditions in mammalian cells. Interestingly, mutations of the major viral fusion protein GP64 that conferred decreased fusogenicity did not affect virus infection of insect cells, whereas virus transduction into mammalian cells was severely impaired, suggesting a more stringent dependence on GP64 fusogenicity for AcMNPV entry into mammalian cells than into insect cells. An increase in the fusogenicity of GP64 mutants resulting from low pH triggered the rescue of fusion-deficient recombinant virus transduction efficiency. Based on the above-described findings, the pH of EEs was specifically reduced with a Na(+)/K(+)-ATPase inhibitor, and the AcMNPV transduction of many mammalian cells indeed became highly efficient. This study not only revealed the roadblocks to mammalian cell entry of baculovirus but also provides a new strategy for improving baculovirus-based gene delivery and therapy. IMPORTANCE Baculoviruses can transduce a wide variety of mammalian cells but do so with low efficiency, which greatly limits their practical application as potential gene delivery vectors. So far, the understanding of baculovirus entry into mammalian cells is obscure, and the limiting stages and factors are unclear. In this study, by comparatively analyzing the mechanisms of baculovirus entry into mammalian and insect cells, virus fusion during the early stage of endocytosis was revealed as the major obstacle for efficient baculovirus transduction into mammalian cells. A higher fusogenicity of the major viral fusion protein GP64 was found to be required for virus entry into mammalian cells than for entry into insect cells. Interestingly, by decreasing the pH of early endosomes with a specific agent, virus transduction of a wide range of mammalian cells was greatly enhanced. This study uncovers the roadblocks to mammalian cell entry of baculoviruses and presents mechanisms to overcome the roadblocks.

Spodoptera litura is an emerging pest insect in cotton and arable crops in Central Asia. To explore the possibility of using baculoviruses as biological control agents instead of chemical pesticides, in a previous study we characterized a number of S. litura nucleopolyhedrovirus (SpltNPV) isolates from Pakistan. We found significant differences in speed of kill, an important property of a biological control agent. Here we set out to understand the genetic basis of these differences in speed of kill, by comparing the genome of the fast-killing SpltNPV-Pak-TAX1 isolate with that of the slow-killing SpltNPV-Pak-BNG isolate. These two isolates and the SpltNPV-G2 reference strain from China were deep sequenced with Illumina. As expected, the two Pakistani isolates were closely related with &gt, 99% sequence identity, whereas the Chinese isolate was more distantly related. We identified two loci that may be associated with the fast action of the SpltNPV-Pak-TAX1 isolate. First, an analysis of rates of synonymous and non-synonymous mutations identified neutral to positive selection on open reading frame (ORF) 122, encoding a viral fibroblast growth factor (vFGF) that is known to affect virulence in other baculoviruses. Second, the homologous repeat region hr17, a putative enhancer of transcription and origin of replication, is absent in SpltNPV-Pak-TAX1 suggesting it may also affect virulence. Additionally, we found there is little genetic variation within both Pakistani isolates, and we identified four genes under positive selection in both isolates that may have played a role in adaptation of SpltNPV to conditions in Central Asia. Our results contribute to the understanding of the enhanced activity of SpltNPV-Pak-TAX1, and may help to select better SpltNPV isolates for the control of S. litura in Pakistan and elsewhere.

gp41 , one of the baculovirus core genes, encodes the only recognized tegument (O-glycosylated) protein of the occlusion-derived virion (ODV) phenotype so far. A previous study using a temperature-sensitive Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) mutant showed that GP41 plays a crucial role in budded virion (BV) formation. However, the precise function of GP41 in the baculovirus replication cycle remains unclear. In this study, AcMNPV GP41 was found to accumulate around the ring zone (RZ) region within the infected nucleus and finally assembled into both BVs and ODVs. Deletion of gp41 from the AcMNPV genome showed that BVs were no longer formed and ODVs were no longer assembled, suggesting the essential role of this gene in baculovirus virion morphogenesis. In infected cells, besides the 42-kDa monomers, dimers and trimers were detected under nonreducing conditions, whereas only trimeric GP41 forms were selectively incorporated into BVs or ODVs. Mutations of all five cysteines in GP41 individually had minor effects on GP41 oligomer formation, albeit certain mutations impaired infectious BV production, suggesting flexibility in the intermolecular disulfide bonding. Single mutations of key leucines within two predicted leucine zipper-like motifs did not interfere with GP41 oligomerization or BV and ODV formation, but double leucine mutations completely blocked oligomerization of GP41 and progeny BV production. In the latter case, the usual subcellular localization, especially RZ accumulation, of GP41 was abolished. The above findings clearly point out a close correlation between GP41 oligomerization and function and therefore highlight the oligomeric state as the functional form of GP41 in the baculovirus replication cycle. IMPORTANCE The tegument, which is sandwiched between the nucleocapsid and the virion envelope, is an important substructure of many enveloped viruses. It is composed of one or more proteins that have important functions during virus entry, replication, assembly, and egress. Unlike another large DNA virus (herpesvirus) that encodes an extensive set of tegument components, baculoviruses very likely exploit the major tegument protein, GP41, to execute functions in baculovirus virion morphogenesis and assembly. However, the function of this O-glycosylated baculovirus tegument protein remains largely unknown. In this study, we identified trimers as the functional structure of GP41 in baculovirus virion morphogenesis and showed that both disulfide bridging and protein-protein interactions via the two leucine zipper-like domains are involved in the formation of different oligomeric states. This study advances our understanding of the unique viral tegument protein GP41 participating in the life cycle of baculoviruses.

Autographa californica nucleopolyhedrovirus (AcMNPV) orf5 (ac5) is a group I alphabaculovirus-specific gene of unknown function, although the protein (AC5) was previously reported to be associated with the per os infectivity factor (PIF) complex. The purpose of this study was to study the dynamics of AC5 during AcMNPV infection and to verify whether it is indeed a component of the PIF complex. Transcription and expression analyses suggested that ac5 is a late viral gene. An ac5-deleted recombinant AcMNPV was generated by homologous recombination. A one-step growth curve assay indicated that ac5 was not required for budded virus (BV) production in Sf9 cells. Scanning electron microscopy and transmission electron microscopy demonstrated that the deletion of ac5 did not affect occlusion body (OB) morphology, and nor did it affect the insertion of occlusion-derived virus (ODV) into OBs. Partially denaturing SDS-PAGE and a co-immunoprecipitation assay clearly showed that AC5 was not a component of the PIF complex, while the deletion of ac5 did not affect the formation and presence of the PIF complex. Further analyses showed, however, that AC5 was an OB-specific protein, but it was not detected as a component of BVs or ODVs. Bioassay experiments showed that the oral infectivity of ac5-deleted AcMNPV to third instar Spodoptera exigua larvae was not significantly different from that of the ac5-repaired virus. In conclusion, AC5 is an intrinsic protein of OBs, instead of being a component of the PIF complex, and is not essential for either BV or ODV infection. AC5 is awaiting the assignment of another hitherto unknown function.

The mosquito-borne chikungunya virus (CHIKV) causes arthritic diseases in humans, whereas the aquatic salmonid alphavirus (SAV) is associated with high mortality in aquaculture of salmon and trout. Using modern biotechnological approaches, promising vaccine candidates based upon highly immunogenic, enveloped virus-like particles (eVLPs) have been developed. However, the eVLP structure (core, lipid membrane, surface glycoproteins) is more complex than that of non-enveloped, protein-only VLPs, which are structurally and morphologically 'simple'. In order to develop an alternative to alphavirus eVLPs, in this paper we engineered recombinant baculovirus vectors to produce high levels of alphavirus core-like particles (CLPs) in insect cells by expression of the CHIKV and SAV capsid proteins. The CLPs localize in dense nuclear bodies within the infected cell nucleus and are purified through a rapid and scalable protocol involving cell lysis, sonication and low-speed centrifugation steps. Furthermore, an immunogenic epitope from the alphavirus E2 glycoprotein can be successfully fused to the N-terminus of the capsid protein without disrupting the CLP self-assembling properties. We propose that immunogenic epitope-tagged alphavirus CLPs produced in insect cells present a simple and perhaps more stable alternative to alphavirus eVLPs.

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.

The entry of baculovirus budded virus into host cells is mediated by two distinct types of envelope fusion proteins (EFPs), GP64 and F protein. Phylogenetic analysis suggested that F proteins were ancestral baculovirus EFPs, whereas GP64 was acquired by progenitor group I alphabaculovirus more recently and may have stimulated the formation of the group I lineage. This study was designed to experimentally recapitulate a possible major step in the evolution of baculoviruses. We demonstrated that the infectivity of an F-null group II alphabaculovirus ( Helicoverpa armigera nucleopolyhedrovirus [HearNPV]) can be functionally rescued by coinsertion of GP64 along with the nonfusogenic F def (furin site mutated HaF) from HearNPV. Interestingly, HearNPV enters cells by endocytosis and, less efficiently, by direct membrane fusion at low pH. However, this recombinant HearNPV coexpressing F def and GP64 mimicked group I virus not only in its EFP composition but also in its abilities to enter host cells via low-pH-triggered direct fusion pathway. Neutralization assays indicated that the nonfusogenic F proteins contribute mainly to binding to susceptible cells, while GP64 contributes to fusion. Coinsertion of GP64 with an F-like protein (Ac23) from group I virus led to efficient rescue of an F-null group II virus. In summary, these recombinant viruses and their entry modes are considered to resemble an evolutionary event of the acquisition of GP64 by an ancestral group I virus and subsequent adaptive inactivation of the original F protein. The study described here provides the first experimental evidence to support the hypothesis of the evolution of baculovirus EFPs.

The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.

Originating from Africa, Usutu virus (USUV) first emerged in Europe in 2001. This mosquito-borne flavivirus caused high mortality rates in its bird reservoirs, which strongly resembled the introduction of West Nile virus (WNV) in 1999 in the United States. Mosquitoes infected with USUV incidentally transmit the virus to other vertebrates, including humans, which can result in neuroinvasive disease. USUV and WNV co-circulate in parts of southern Europe, but the distribution of USUV extends into central and northwestern Europe. In the field, both viruses have been detected in the northern house mosquito Culex pipiens, of which the potential for USUV transmission is unknown. To understand the transmission dynamics and assess the potential spread of USUV, we determined the vector competence of C. pipiens for USUV and compared it with the well characterized WNV. We show for the first time that northwestern European mosquitoes are highly effective vectors for USUV, with infection rates of 11% at 18 °C and 53% at 23 °C, which are comparable with values obtained for WNV. Interestingly, at a high temperature of 28 °C, mosquitoes became more effectively infected with USUV (90%) than with WNV (58%), which could be attributed to barriers in the mosquito midgut. Small RNA deep sequencing of infected mosquitoes showed for both viruses a strong bias for 21-nucleotide small interfering (si)RNAs, which map across the entire viral genome both on the sense and antisense strand. No evidence for viral PIWI-associated RNA (piRNA) was found, suggesting that the siRNA pathway is the major small RNA pathway that targets USUV and WNV infection in C. pipiens mosquitoes., Graphical abstract, Highlights • Northwestern European mosquitoes are highly effective vectors for USUV. • Culex pipiens is significantly more competent for USUV than for WNV at 28 °C. • The siRNA but not the piRNA pathway targets USUV and WNV infections in C. pipiens. • USUV may be a prelude to WNV transmission in northwestern Europe.

Chikungunya virus nonstructural protein nsP3 has an essential but unknown role in alphavirus replication and interacts with Ras-GAP SH3 domain-binding protein (G3BP). Here we describe the first known function of nsP3, to inhibit stress granule assembly by recruiting G3BP into cytoplasmic foci. A conserved SH3 domain-binding motif in nsP3 is essential for both nsP3-G3BP interactions and viral RNA replication. This study reveals a novel role for nsP3 as a regulator of the cellular stress response.

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae ) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.

Baculoviruses induce apoptotic responses in cultured insect cells, which can severely limit viral replication. To overcome this host response baculoviruses carry anti-apoptotic genes, including members of the p35 and inhibitor of apoptosis (iap) gene families. The baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV) carries two putative apoptosis suppressor genes (iap2 and iap3), which we studied in more detail. IAPs are believed to be functional in the cytoplasm, but surprisingly, when transiently expressed as EGFP fusions, IAP2 was evenly distributed throughout the cell, while IAP3 was mainly found in the nucleus. The latter became evenly distributed in both compartments in HearNPV infected cells. When iap2 was deleted, HearNPV could be propagated in Hz2e5 cells, while an iap3 deletion was lethal. The HearNPV Δiap3 mutant could be rescued by reinsertion of the HearNPV iap3 gene and by the well-studied anti-apoptotic genes Autographa californica (Ac)MNPV p35 or Orgyia pseudotsugata (Op)MNPV iap3. RNAi analysis showed that HearNPV induced apoptosis in Hz2e5 cells transfected with iap3 dsRNA, while silencing of iap2 did not lead to apoptosis. Finally, IAP3 was able to inhibit actinomycin-D induced apoptosis when transiently expressed in Sf21 cells. These results together indicate that HearNPV IAP3 is a functional apoptosis suppressor, while the function of IAP2 remains elusive.

ORF100 (ha100) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been reported as one of the unique genes of group II alphabaculoviruses encoding a protein located in the occlusion-derived virus (ODV) envelope and nucleocapsid. The protein consists of 510 aa with a predicted mass of 58.1 kDa and is a homologue of poly(ADP–ribose) glycohydrolase in eukaryotes. Western blot analysis detected a 60 kDa band in HearNPV-infected HzAM1 cells starting at 18 h post-infection. Transient expression of GFP-fused HA100 in HzAM1 cells resulted in cytoplasmic localization of the protein, but after superinfection with HearNPV, GFP-fused HA100 was localized in the nucleus. To study the function of HA100 further, an ha100-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the ha100-null virus had similar budded virus production kinetics to that of the parental virus. Electron microscopy revealed that deletion of HA100 did not alter the morphology of ODVs or occlusion bodies (OBs). However, bioassays in larvae showed that the 50 % lethal concentration (LC50) value of HA100-null OBs was significantly higher than that of parental OBs; the median lethal time (LT50) of ha100-null OBs was about 24 h later than control virus. These results indicate that HA100 is not essential for virus replication in vitro. However, it significantly affects the oral infectivity of OBs in host insects, suggesting that the association HA100 with the ODV contributes to the infectivity of OBs in vivo.

Cyclobutane pyrimidine dimer (CPD) photolyases repair ultraviolet (UV)-induced DNA damage using blue light. To get insight in the origin of baculovirus CPD photolyase (phr) genes, homologues in the lepidopteran insects Chrysodeixis chalcites, Spodoptera exigua and Trichoplusia ni were identified and characterized. Lepidopteran and baculovirus phr genes each form a monophyletic group, and together form a well-supported clade within the insect photolyases. This suggests that baculoviruses obtained their phr genes from an ancestral lepidopteran insect host. A likely evolutionary scenario is that a granulovirus, Spodoptera litura GV or a direct ancestor, obtained a phr gene. Subsequently, it was horizontally transferred from this granulovirus to several group II nucleopolyhedroviruses (NPVs), including those that infect noctuids of the Plusiinae subfamily.

Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% β-mercaptoethanol with heating at 50°C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection.

Naturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus-killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild-type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild-type and one of the recombinant viruses. An egt-negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt-deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT-(Androctonus australis Hector) insect-selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT-expressing recombinant and the wild-type virus died at positions significantly lower, compared to infection with the pure wild-type viral strain. The results indicate that transmission of egt-negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild-type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment.

Cyclobutane pyrimidine dimer (CPD) photolyases convert UV-induced CPDs in DNA into monomers using visible light as the energy source. Two phr genes encoding class II CPD photolyases PHR1 and PHR2 have been identified in Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV). Transient expression assays in insect cells showed that PHR1-EGFP fusion protein was localized in the nucleus. Early after transfection, PHR2-EGFP was distributed over the cytoplasm and nucleus but, over time, it became localized predominantly in the nucleus. Immunofluorescence analysis with anti-PHR2 antiserum showed that, early after transfection, non-fused PHR2 was already present mainly in the nucleus, suggesting that the fusion of PHR2 to EGFP hindered its nuclear import. Both PHR-EGFP fusion proteins strongly colocalized with chromosomes and spindle, aster and midbody structures during host-cell mitosis. When PHR2-EGFP-transfected cells were superinfected with Autographa californica multiple-nucleocapsid NPV (AcMNPV), the protein colocalized with virogenic stroma, the replication factories of baculovirus DNA. The collective data support the supposition that the PHR2 protein plays a role in baculovirus DNA repair.

Fast-acting recombinant baculoviruses have potential for improved insect pest suppression. However, the ecological impact of using such viruses must be given careful consideration. One strategy for mitigating risks might be simultaneous release of a wild-type baculovirus, so as to facilitate rapid displacement of the recombinant baculovirus by a wild-type. However, at what ratio must the two baculoviruses be released? An optimum release ratio must ensure both fast action, and the eventual competitive displacement of the recombinant virus and fixation of the wild-type baculovirus in the insect population. Here we challenged Trichoplusia ni larvae with different ratios of wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and a derived recombinant, vEGTDEL, which has the endogenous egt gene (coding for ecdysteroid UDP-glucosyltransferase) deleted. Time to death increased with the proportion wild-type virus in the inoculum mixture, although a 1:10 ratio (wild-type: recombinant) resulted in equally rapid insecticidal action as vEGTDEL alone. Five serial passages of three different occlusion body (OB) mixtures of the two viruses were also performed. OBs from 10 larval cadavers were pooled and used to initiate the following passage. Although the wild-type baculovirus was maintained over five passages, it did not go to fixation in most replicates of the serial passage experiment (SPE), and there was no good evidence for selection against the recombinant. Long-term maintenance of a recombinant in serial passage suggests an ecosystem safety risk. We conclude that for assessing ecological impact of recombinant viruses, SPEs in single and multiple larvae are relevant because of potential modulating effects at the between-host level.

The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been performed to elucidate putative functions of the annotated open reading frames of this virus and this endeavour is still ongoing. AcMNPV is the most well-known and well-studied baculovirus species, not in the least for its application as a vector for the high-level expression of foreign genes in insect cells. This article is the first monograph of a single baculovirus and gives a current overview of what is known about the 151 AcMNPV ORFs, including (putative) function and temporal and spatial presence of transcripts and protein. To date 60 ORFs have a proven function, another 19 ORFs have homologs for which functions are known in other baculoviruses and 72 ORFs are still enigmatic. This paper should assist the reader in quickly finding the essentials of AcMNPV.

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.

Programmed cell death or apoptosis is a major defense mechanism in insects in response to viral infections. The genome of Chilo iridescent virus (CIV) has three ORFs with homology to baculovirus inhibitor of apoptosis (iap) genes. The proteins encoded by the 157L, 193R, and 332L ORFs contain 152, 208 and 234 amino acids, respectively. While all three proteins contain C-terminal RING domains, only the protein encoded by ORF 193R contains a baculoviral iap repeat (BIR) domain, indicative of a putative IAP protein. The 193R protein has 28 and 27% similarity in amino acid sequence to the Orgyia pseudotsugata MNPV and Cydia pomonella granulovirus IAP-3 proteins, respectively. ORF 193R from CIV is the only gene known to exist among the Iridoviridae that encodes a BIR domain. 193R is transcribed early during CIV infection, and its transcription is not dependent on the synthesis of early viral proteins. When this putative CIV IAP was transiently expressed in SPC-BM-36 and Sf21 cells under the control of an immediate early baculovirus promoter it significantly reduced apoptosis induced by actinomycin-D. Silencing of the CIV iap gene (193R) in CIV infected SPC-BM-36 cells with 193R-specific dsRNA resulted in apoptosis. Thus, CIV ORF 193R is the first iap gene identified in an iridovirus, which encodes a functional IAP protein.

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.

The open reading frame Ha107 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) encodes a putative protein of 51 kDa with homologues in a few group II NPVs and a granulovirus. Ha107 was transcribed as polyadenylated transcripts in infected HzAM1 insect cells. The transcripts were initiated at two distinct locations, one upstream of Ha106 (superoxide dismutase gene, sod) and the second upstream of Ha107. By Western blot analysis, two forms of the HA107 protein were detected in infected cells, a major polypeptide of 48 kDa and a minor one of 51 kDa. Western blot and immunoelectron microscopy analyses further showed that the HA107 protein was associated with the nucleocapsids of both budded virions (BVs) and occlusion-derived virions. A Ha107 knockout virus expressing enhanced green fluorescent protein and polyhedrin was constructed using bacmid technology. A one-step virus growth curve indicated that the BV titre of the knockout virus was significantly higher than that of the parental virus and a Ha107 repair virus. Bioassays indicated that the knockout virus was able to infect third-instar H. armigera larvae; however, its median lethal dose (LD50) was significantly higher than those of the parental virus and Ha107 repair virus. These data indicate that Ha107 encodes a non-essential structural protein of HearNPV virions and that deletion of this gene increases the BV titre and LD50 of the occluded virus.

The Douglas-fir tussock moth Orgyia pseudotsugata (Lepidoptera: Lymantriidae) is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus (SNPV) isolated from O. pseudotsugata larvae in Canada (OpSNPV) was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group II NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for

Highlights • Baculovirus budded virion ultrastructure has been revisited using cryo-electron microscopy. • The now well-preserved virions have a remarkable elongated, ovoid shape and large lateral space between nucleocapsid and the intact envelope. • Consistent with previous findings using classical electron microscopy the nucleocapsid has a distinctive cap and base structure interacting tightly with the envelope. • Most spikes are densely clustered at the two apical ends of the virion. Using cryo-electron microscopy the viral envelope appeared to contain two layers with a total thickness of ≈6–7 nm, which is significantly thicker than a usual biological membrane (, Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈6–7 nm, which is significantly thicker than a usual biological membrane (

Many parasites manipulate host behaviour to enhance parasite transmission and survival. A fascinating example is baculoviruses, which often induce death in caterpillar hosts at elevated positions ('tree-top' disease). To date, little is known about the underlying processes leading to this adaptive host manipulation. Here, we show that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) triggers a positive phototactic response in S. exigua larvae prior to death and causes the caterpillars to die at elevated positions. This light-dependent climbing behaviour is specific for infected larvae, as movement of uninfected caterpillars during larval development was light-independent. We hypothesize that upon infection, SeMNPV captures a host pathway involved in phototaxis and/or light perception to induce this remarkable behavioural change.

BACKGROUND: Chikungunya virus (CHIKV) is an arthritogenic alphavirus (family Togaviridae), transmitted by Aedes species mosquitoes. CHIKV re-emerged in 2004 with multiple outbreaks worldwide and recently reached the Americas where it has infected over a million individuals in a rapidly expanding epidemic. While alphavirus replication is well understood in general, the specific function (s) of non-structural protein nsP3 remain elusive. CHIKV nsP3 modulates the mammalian stress response by preventing stress granule formation through sequestration of G3BP. In mosquitoes, nsP3 is a determinant of vector specificity, but its functional interaction with mosquito proteins is unclear. METHODS: In this research we studied the domains required for localization of CHIKV nsP3 in insect cells and demonstrated its molecular interaction with Rasputin (Rin), the mosquito homologue of G3BP. The biological involvement of Rin in CHIKV infection was investigated in live Ae. albopictus mosquitoes. RESULTS: In insect cells, nsP3 localized as cytoplasmic granules, which was dependent on the central domain and the C-terminal variable region but independent of the N-terminal macrodomain. Ae. albopictus Rin displayed a diffuse, cytoplasmic localization, but was effectively sequestered into nsP3-granules upon nsP3 co-expression. Site-directed mutagenesis showed that the Rin-nsP3 interaction involved the NTF2-like domain of Rin and two conserved TFGD repeats in the C-terminal variable domain of nsP3. Although in vitro silencing of Rin did not impact nsP3 localization or CHIKV replication in cell culture, Rin depletion in vivo significantly decreased the CHIKV infection rate and transmissibility in Ae.albopictus. CONCLUSIONS: We identified the nsP3 hypervariable C-terminal domain as a critical factor for granular localization and sequestration of mosquito Rin. Our study offers novel insight into a conserved virus-mosquito interaction at the molecular level, and reveals a strong proviral role for G3BP homologue Rin in live mosquitoes, making the nsP3-Rin interaction a putative target to interfere with the CHIKV transmission cycle.

Background West Nile virus (WNV) is a highly pathogenic flavivirus transmitted by Culex spp. mosquitoes. In North America (NA), lineage 1 WNV caused the largest outbreak of neuroinvasive disease to date, while a novel pathogenic lineage 2 strain circulates in southern Europe. To estimate WNV lineage 2 epidemic potential it is paramount to know if mosquitoes from currently WNV-free areas can support further spread of this epidemic. Methodology/Principal Findings We assessed WNV vector competence of Culex pipiens mosquitoes originating from north-western Europe (NWE) in direct comparison with those from NA. We exposed mosquitoes to infectious blood meals of lineage 1 or 2 WNV and determined the infection and transmission rates. We explored reasons for vector competence differences by comparing intrathoracic injection versus blood meal infection, and we investigated the influence of temperature. We found that NWE mosquitoes are highly competent for both WNV lineages, with transmission rates up to 25%. Compared to NA mosquitoes, transmission rates for lineage 2 WNV were significantly elevated in NWE mosquitoes due to better virus dissemination from the midgut and a shorter extrinsic incubation time. WNV infection rates further increased with temperature increase. Conclusions/Significance Our study provides experimental evidence to indicate markedly different risk levels between both continents for lineage 2 WNV transmission and suggests a degree of genotype-genotype specificity in the interaction between virus and vector. Our experiments with varying temperatures explain the current localized WNV activity in southern Europe, yet imply further epidemic spread throughout NWE during periods with favourable climatic conditions. This emphasizes the need for intensified surveillance of virus activity in current WNV disease-free regions and warrants increased awareness in clinics throughout Europe., Author Summary West Nile virus (WNV) is on the rise in Europe, with increasing numbers of human cases of neurological disease and death since 2010. However, it is currently unknown whether or not WNV will continue to spread to north-western Europe (NWE), in a fashion similar to the WNV epidemic sweep in the United States (1999–2004). The presence of competent mosquitoes is a strict requirement for WNV transmission, but no laboratory studies have been conducted with the new European lineage 2 WNV outbreak strain. Our study is the first to investigate transmissibility in NWE Culex pipiens for lineage 2 WNV in a systematic, direct comparison with North American Culex pipiens and with the lineage 1 WNV strain. We demonstrate that European mosquitoes are highly competent for both WNV lineages, which underscores the epidemic potential of WNV in Europe. However, the transmission rate for lineage 2 WNV was significantly lower in North American mosquitoes, which indicates different risk levels between both continents for lineage 2 but not lineage 1 WNV. Based on our result, we propose that WNV surveillance in mosquitoes and birds must be intensified in Europe to allow early detection, timely intervention strategies and prevent outbreaks of WNV neurological disease.

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.

Entry of the budded virus form of baculoviruses into insect and mammalian cells is generally thought to occur through a low-pH-dependent endocytosis pathway, possibly through clathrin-coated pits. This insight is primarily based on (immuno)electron microscopy studies but requires biochemical support to exclude the use of other pathways. Here, we demonstrate using various inhibitors that functional entry of baculoviruses into insect and mammalian cells is primarily dependent on clathrin-mediated endocytosis. Our results further suggest that caveolae are somehow involved in baculovirus entry in mammalian cells. A caveolar endocytosis inhibitor, genistein, enhances baculovirus transduction in these cells considerably.

Open reading frame 132 (Ha132) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is a homologue of per os infectivity factor 2 (pif-2) of Spodoptera exigua multiple nucleopolyhedrovirus. Sequence analysis indicated that Ha132 encoded a protein of 383 aa with a predicted molecular mass of 44.5 kDa. Alignment of HA132 and its baculovirus homologues revealed that HA132 was highly conserved among baculoviruses, with 14 absolutely conserved cysteine residues. RT-PCR indicated that Ha132 was first transcribed at 24 h post-infection. Western blot analysis showed that a 43 kDa band was detectable in HearNPV-infected HzAM1 cells from 36 h post-infection. Western blots also indicated that HA132 was a component of the occlusion-derived virus, but not of budded virus. Deletion of Ha132 from HearNPV abolished per os infectivity, but had no effect on the infectivity of the budded virus phenotype.

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.

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 fp25k gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was studied. HearNPV fp25k gene transcription was found starting from about 18 h post-infection, and protein could be detected from the same time with antiserum against FP25K. To study the function of HearNPV fp25k, a recombinant HearNPV (HaBacWD11) with an enhanced green fluorescent protein (GFP) gene replacing the fp25k was constructed using HaBacHZ8, a bacmid of HearNPV that lacks the polyhedrin gene. Growth curve analysis showed that HaBacWD11 produced higher titres of budded viruses (BVs) than its wild-type counterpart HaBacHZ8–GFP. Electron microscopic analysis indicated that at the late stage of infection, the number of intranuclear enveloped nucleocapsids in HaBacWD11-infected cells was much less than that of HaBacHZ8–GFP. A rescue recombinant virus HaBacWD14 was constructed by reintroducing fp25k gene into HaBacWD11. The growth curve and electron microscopic analysis of the rescued recombinant confirmed that the increase of BV yield and the decrease of the virion production in infected cells were the result of fp25k deletion. The expression of membrane fusion protein (Ha133) and ODV-E66 were studied using the FP25K mutants HaBacWD11 and HaBacHZ8–GFP. Unlike FP25K mutants in Autographa californica multicapsid NPV (AcMNPV), which caused an increase in the expression of membrane fusion protein GP64 and a decrease of ODV-E66, no obvious changes at the expression level of Ha133 and ODV-E66 were observed in HearNPV FP25K mutant.

The complete genome sequence of a single nucleocapsid nucleopolyhedrovirus recently isolated from Chrysodeixis chalcites (ChchNPV) was determined. The viral genome has a size of 149 622 bp and an overall G+C content of 39·1 mol%. The sequence contains 151 predicted open reading frames (ORFs) with a minimal size of 50 codons. The similarity of these ORFs with those of other completely sequenced baculoviruses was calculated using a newly developed database, named gecco. Phylogenetic analysis of the whole genome confirmed the evolutionary relationship of ChchNPV with group II NPVs, as did the absence of the NPV group I-specific gp64 gene. It is the first group II NPV to encode proliferating cell nuclear antigen. Most noteworthy is the presence of two ORFs encoding a class II cyclobutane pyrimidine dimer DNA photolyase. These two ORFs share only 45 % amino acid identity and have different promoter motifs. Twenty-two additional unique baculovirus genes were identified, including a gene encoding a novel putative RING finger protein with a possible homologue in poxviruses.

Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67deltaSS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculovirus envelope protein GP64. Both GFP:p67deltaSS and GP64:p67C induced antibodies with high ELISA titers that neutralized T. parva sporozoites with high efficiency. Upon challenge, a correlation was observed between the in vitro neutralizing capacity and the reduction in severe ECF for individual animals. A protection level upto 85% was obtained. This level of protection was achieved with only two inoculations of 100 microg per dose, which is a major improvement over previous recombinant p67 products.

A hitherto unknown single nucleocapsid nucleopolyhedrovirus (SNPV) with a unique property was isolated from larvae of the looper Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). Polyhedrin, lef-8, and pif-2 gene sequences were obtained by PCR with degenerate primers and used for phylogenetic analysis. ChchNPV belonged to class II NPVs and its polyhedrin sequence was most similar to that of class II NPVs of other members of the subfamily Plusiinae. Further genetic characterization involved the random cloning of HindIII fragments into a plasmid vector and analysis by end-in sequencing. A gene so far unique to baculoviruses was identified, which encodes a putative DNA repair enzyme: cyclobutane pyrimidine dimer (CPD) DNA photolyase (dpl). The transcriptional activity of this gene was demonstrated in both ChchNPV-infected C. chalcites larvae and infected Trichoplusia W High Five cells by RT-PCR and 5' and 3' RACE analysis. The possible role of this gene in the biology of the virus is discussed. (C) 2004 Elsevier Inc. All rights reserved.

Continuous protein production with baculovirus expression vectors in insect-cell bioreactors is characterized by a dramatic drop in heterologous protein production within a few weeks. This is mainly due to the spontaneous deletion of the heterologous gene(s) from the baculovirus genome and/or to the rapid accumulation of defective interfering baculoviruses (DIs). Cell culture experiments with bacmid-derived baculoviruses showed that spontaneous deletions in the foreign bacterial artificial chromosome (BAC) sequences readily occurred, These deletions correlated with a low density of baculovirus homologous (repeat) regions (hrs), which are located dispersed throughout the baculovirus genome and are believed to act as origins of viral DNA replication (oris). To test the hypothesis that deletions are more likely to occur in regions with a low ori density, the properties of bacmidderived baculoviruses with an additional hr in the unstable BAC sequences were compared to the standard bacmidderived baculovirus in a continuous cascaded insect-cell bioreactor configuration. All viruses were equipped with a green fluorescent protein (GFP) gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). The insertion of an extra hr in the BAC vector led to improved genetic stability of adjacent sequences, resulting in prolonged protein expression. The maintenance of the BAC sequences appeared to be dependent on the orientation of the inserted hr. The advantages of the utilization of hrs to improve the stability of baculovirus expression vectors for the large-scale protein production in insect-cell bioreactors are discussed. (C) 2004 Wiley Periodicals, Inc.

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.

The Spodoptera exigua multicapsid nucleopolyhedrovirus (Se M NPV) Se8 gene was recently shown to encode the viral envelope fusion (F) protein. A 60-kDa C-terminal subunit (F 1 ) of the 76-kDa primary translation product of this gene was found to be the major envelope protein of Se M NPV budded virus (BV) (W. F. J. IJkel, M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema, Virology 275:30–41, 2000). A specific inhibitor was used to show that furin is involved in cleavage of the precursor envelope fusion (F 0 ) protein. BV produced in the presence of the inhibitor possesses the uncleaved F 0 protein, while an F protein with a mutation in the furin cleavage site was translocated to the plasma membrane but lost its fusogenic activity. These results indicate that cleavage of F 0 is required to activate the Se M NPV F protein and is necessary for BV infectivity. Specific antibodies against F 1 and against the putative N terminus (F 2 ) of the primary translation product were used to show that the F protein is BV specific and that BVs contain both the 60- (F 1 ) and 21-kDa (F 2 ) cleavage products. In nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis both subunits migrate as a single 80-kDa protein, indicating that the subunits remain associated by a disulfide linkage. In addition, the presence of the F protein predominately as a monomer suggests that disulfide links are not involved in oligomerization. Thus, the envelope fusion protein from group II nucleopolyhedroviruses of baculoviruses has properties similar to those of proteins from a number of vertebrate viruses.

Scenario studies were carried out with a process-based model for control of wild-type and genetically modified baculoviruses in populations of Spodoptera exigua in glasshouse chrysanthemum (BACSIM). These scenario studies were used to evaluate the effectiveness of different spraying regimes, concentrations, UV-protection agents, and mean time to kill insect. In simulations on concentration and timing of S. exigua multicapsid nucleopolyhedrovirus (SeMNPV), good control efficacy was obtained with SeMNPV concentrations of 1×107 polyhedra/m2 or higher. An early timing of virus applications, soon after egg deposition, is essential for effective control, especially at high temperatures, when the developmental rate of larvae is high and the window of opportunity for virus control is short. UV-protection agents contribute only marginally to effective biological control in the glasshouse because the decay rates of SeMNPV and Autographa californica multicapsid nucleopolyhedrovirus already ensure a period of exposure to active virus that is long enough for caterpillars to acquire a lethal concentration under practical conditions. The effect of genetic improvement towards shortening the survival time of infected larvae depends on the ability to time virus applications accurately and the mean time to kill the unmodified virus. Recombinant viruses with a lower mean time to kill are more effective than wild-type viruses when applied against second, third, and fourth instars because in these stages S. exigua infests a considerable number of plants and the rate of foliage consumption increases rapidly. When applied against first instars that infest only a small number of plants and have low consumption rates, recombinants will only be slightly more effective than wild-type viruses. In the case of an unmodified virus that already has a relatively high mean time to kill, a further reduction in the time to kill will not result in improved control. Thus, accurate monitoring followed by virus application is indispensable for adequate control and may even alleviate the need for fast killing viruses.

Salmonid alphavirus (SAV; also known as Salmon pancreas disease virus; family Togaviridae) causes pancreas disease and sleeping disease in Atlantic salmon and rainbow trout, respectively, and poses a major burden to the aquaculture industry. SAV infection in vivo is temperature-restricted and progeny virus is only produced at low temperatures (10–15 °C). Using engineered SAV replicons we show that viral RNA replication is not temperature-restricted suggesting that the viral structural proteins determine low-temperature dependency. The processing/trafficking of SAV glycoproteins E1 and E2 as a function of temperature was investigated via baculovirus vectors in Sf9 insect cells and by transfection of CHSE-214 fish cells with DNA constructs expressing E1 and E2. We identified SAV E2 as the temperature determinant by demonstrating that membrane trafficking and surface expression of E2 occurs only at low temperature and only in the presence of E1. Finally, a vaccination-challenge model in Atlantic salmon demonstrates the biological significance of our findings and shows that SAV replicon DNA vaccines encoding E2 elicit protective immunity only when E1 is co-expressed. This is the first study that identifies E2 as the critical determinant of SAV low-temperature dependent virion formation and defines the prerequisites for induction of a potent immune response in Atlantic salmon by DNA vaccination.

White spot syndrome virus (WSSV) is a large DNA virus infecting shrimp and other crustaceans. The virus particles contain at least five major virion proteins, of which three (VP26, VP24, and VP15) are present in the rod-shaped nucleocapsid and two (VP28 and VP19) reside in the envelope. The mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon, and the role of these proteins in these processes are not known. A specific polyclonal antibody was generated against the major envelope protein VP28 using a baculovirus expression vector system. The VP28 antiserum was able to neutralize WSSV infection of P. monodon in a concentration-dependent manner upon intramuscular injection. This result suggests that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic WSSV infection in shrimp.