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The standard fermentor has been and is the workhorse of the bioreactor stable used in biotechnology. The effects of hydrodynamic forces on animal cells in a stirred bioreactor have been most extensively studied in microcarrier systems, where the cells grow attached to the surface of spherical particles typically about 180 µm in diameter. Insect cells and animal cells can be grown well in large vessels. The Kolmogorov theory and the hypothetical killing volume model are, in this respect, the promising approaches for optimal design of large-scale animal cell bioreactors. In addition, sparged gas bubbles subject the cell to surface tension forces and to fluid mechanical forces resulting from the motion, disengagement and bursting of bubbles, and from foaming. Animal cells on microcarriers are especially susceptible to shear. In addition to the lack of a protective cell wall and their relatively large size, they also lack individual cell mobility.

EFSA is requested to assess the safety of a broad range of biological agents in the context of notification for market authorisation as sources of food and feed additives, food enzymes and plant protection products. The qualified presumption of safety (QPS) assessment was developed to provide a harmonised generic pre‐assessment to support safety risk assessments performed by EFSA's scientific Panels. The safety of unambiguously defined biological agents (at the highest taxonomic unit appropriate for the purpose for which an application is intended), and the completeness of the body of knowledge are assessed. Identified safety concerns for a taxonomic unit are, where possible and reasonable in number, reflected as ‘qualifications’ in connection with a recommendation for a QPS status. The list of QPS recommended biological agents was reviewed and updated in the current opinion and therefore becomes the valid list. The 2016 update reviews previously assessed microorganisms including bacteria, yeasts and viruses used for plant protection purposes following an Extensive Literature Search strategy. The taxonomic units related to the new notifications received since the 2013 QPS opinion, were periodically evaluated for a QPS status and the results published as Statements of the BIOHAZ Panel. Carnobacterium divergens, Lactobacillus diolivorans, Microbacterium imperiale, Pasteuria nishizawae, Pediococcus parvulus, Bacillus flexus, Bacillus smithii, Xanthomonas campestris and Candida cylindracea were recommended for the QPS list. All taxonomic units previously recommended for the 2013 QPS list had their status reconfirmed as well their qualifications with the exception of Pasteuria nishizawae for which the qualification was removed. The exclusion of filamentous fungi and enterococci from the QPS evaluations was reconsidered but monitoring will be maintained and the status will be re‐evaluated in the next QPS Opinion update. Evaluation of bacteriophages should remain as a case‐by‐case procedure and should not be considered for QPS status., This publication is linked to the following EFSA Journal article: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2017.4663/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2017.e15031/full

Previously, we observed that the transcripts of Invertebrate iridescent virus 6 (IIV6) are not polyadenylated, in line with the absence of canonical poly(A) motifs (AATAAA) downstream of the open reading frames (ORFs) in the genome. Here, we determined the 3' ends of the transcripts of fifty-four IIV6 virion protein genes in infected Drosophila Schneider 2 (52) cells. By using ligation-based amplification of cDNA ends (LACE) it was shown that the IIV6 mRNAs often ended with a CAUUA motif. In silico analysis showed that the 3'-untranslated regions of IIV6 genes have the ability to form hairpin structures (22-56 nt in length) and that for about half of all IIV6 genes these 3' sequences contained complementary TAATG and CATTA motifs. We also show that a hairpin in the 3' flanking region with conserved sequence motifs is a conserved feature in invertebrate-infecting iridoviruses (genus Iridovirus and Chloriridovirus).

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.

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 >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.

Microorganisms are intentionally added at different stages of the food and feed chain (food or feed additive, novel food or plant protection product) and are subjected to regulation and safety assessment by the European Food Safety Authority. Safety evaluation is based on application dossiers for market authorisation to the European Commission. The qualified presumption of safety (QPS) concept was developed in 20031 to provide a harmonised generic safety pre-appraisal of the above microorganisms. Unambiguously defined biological taxonomic units (TUs) are assessed for their body of knowledge, their safety and their end use. Identified safety concerns for a certain TU can be, where reasonable in number and not universally present, reflected as ‘qualifications.’ Strains belonging to TUs having QPS status may benefit of a fast track evaluation. The lowest TU for which the QPS status is granted is the species level for bacteria and yeasts and the family for viruses. The QPS concept is also applicable to genetically modified microorganisms used for production purposes. Based on the current body of knowledge and/or the ambiguous taxonomic position, some TUs, such as filamentous fungi, bacteriophages, Enterococcus faecium, Escherichia coli, Streptomyces spp. and Oomycetes, are not considered liable for QPS status., The qualified presumption of safety assessment and its role in EFSA risk evaluations: 15 years past.

Previously we have generated a recombinant Chilo iridescent virus (CIV) by inserting the green fluorescent protein gene (gfp) into the CIV 157L open reading frame (ORF) locus and showed that this recombinant (rCIV-Delta 157L-gfp) was fully infectious both in cell culture as well as in insect larvae. This study opened up a new avenue for increasing the speed of kill of CIV and other iridoviruses by inserting virulence or toxin genes into the viral genome. In the current study we constructed a recombinant CIV (rCIV-Delta 157L/gfp-AaIT) where the 157L ORF was replaced with both the AaIT neurotoxin gene from the scorpion Androctonus australis and the gfp gene, each under control of the viral major capsid protein (mcp) gene promoter. Recombinant virus was purified by successive rounds of plaque purification using Spodoptera frugiperda (Sf-9) cells. One-step growth curves for the recombinant viruses, rCIV-Delta 157L/gfp-AaIT and rCIV-Delta 157L-gfp, and wild-type CIVs in Sf-9 cells showed similar profiles. AaIT toxin expression in infected third instar Galleria mellonella larvae was confirmed by western blot analysis using an antibody against the AaIT protein. rCIV-Delta 157L/gfp-AaIT infection at a concentration that kills 100% of the larvae caused paralysis in infected third instar G. mellonella larvae from two days after injection, whereas infection with non-AaIT containing viruses showed mortality starting much later (>10 days). Bioassays on these larvae demonstrated that the speed of kill of CIV carrying AaIT was strikingly enhanced as compared to wild-type CIV. These results suggest that insertion of a toxin gene into CIV provides further opportunities to control a wide range of pest insects, such as weevils, using an iridovirus. (C) 2016 Elsevier Inc. All rights reserved.

Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of ∼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an ∼400-kDa subcomplex. Deletion of pif0, -4, -6, -7, or -9 resulted in a subcomplex of ∼230 kDa, but deletion of pif1, -2, or -3 abolished formation of any complex. Taken together, our data identified a core complex of ∼230 kDa, consisting of PIF1, -2, and -3. This revised the previous knowledge that the core complex was about 170 kDa and contained PIF1 to -4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection. IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to ∼230 kDa and consists of PIF1 to -3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODVs. Only the full complex is resistant to proteolytic degradation in the insect midgut, implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.

Background The management of the tsetse species Glossina pallidipes (Diptera; Glossinidae) in Africa by the sterile insect technique (SIT) has been hindered by infections of G. pallidipes production colonies with Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae family). This virus can significantly decrease productivity of the G. pallidipes colonies. Here, we used three highly diverged genes and two variable number tandem repeat regions (VNTRs) of the GpSGHV genome to identify the viral haplotypes in seven Glossina species obtained from 29 African locations and determine their phylogenetic relatedness. Results GpSGHV was detected in all analysed Glossina species using PCR. The highest GpSGHV prevalence was found in G. pallidipes colonized at FAO/IAEA Insect Pest Control Laboratory (IPCL) that originated from Uganda (100%) and Tanzania (88%), and a lower prevalence in G. morsitans morsitans from Tanzania (58%) and Zimbabwe (20%). Whereas GpSGHV was detected in 25–40% of G. fuscipes fuscipes in eastern Uganda, the virus was not detected in specimens of neighboring western Kenya. Most of the identified 15 haplotypes were restricted to specific Glossina species in distinct locations. Seven haplotypes were found exclusively in G. pallidipes. The reference haplotype H1 (GpSGHV-Uga; Ugandan strain) was the most widely distributed, but was not found in G. swynnertoni GpSGHV. The 15 haplotypes clustered into three distinct phylogenetic clades, the largest contained seven haplotypes, which were detected in six Glossina species. The G. pallidipes-infecting haplotypes H10, H11 and H12 (from Kenya) clustered with H7 (from Ethiopia), which presumably corresponds to the recently sequenced GpSGHV-Eth (Ethiopian) strain. These four haplotypes diverged the most from the reference H1 (GpSGHV-Uga). Haplotypes H1, H5 and H14 formed three main genealogy hubs, potentially representing the ancestors of the 15 haplotypes. Conclusion These data identify G. pallidipes as a significant driver for the generation and diversity of GpSGHV variants. This information may provide control guidance when new tsetse colonies are established and hence, for improved management of the virus in tsetse rearing facilities that maintain multiple Glossina species. Electronic supplementary material The online version of this article (10.1186/s12866-018-1297-2) contains supplementary material, which is available to authorized users.

Background Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160–174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV’s specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV’s asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models. Results Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host’s immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly’s RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly. Conclusions The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive ‘explorers’ to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

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.

Spodoptera litura is an emerging insect pest in a wide range of crops worldwide. The insect is difficult to control because of resistance development to synthetic insecticides and emerging resistance to Bacillus thuringiensis toxins. Therefore, there is a need to develop biological control agents, preferably from an indigenous source to avoid risks associated with the importation of exotic natural antagonists. A Pakistani isolate of S. litura nucleopolyhedrovirus (SpltNPV, Baculoviridae), SpltNPV-Pak-BNG, was obtained from the field and characterized biologically and genetically, and compared to a SpltNPV reference isolate, SpltNPV-G1, thought to be of Chinese origin. The dose–mortality response (LD50) of SpltNPV-Pak-BNG was not significantly different from that of the reference isolate SpltNPV-G1, but the time-to-death (LT50) was significantly shorter for SpltNPV-Pak-BNG than for SpltNPV-G1. DNA restriction enzyme profiling indicated that SpltNPV-Pak-BNG and SpltNPV-G1 are different viruses. Sequence analysis of ‘ORF24’, specific for SpltNPV (and S. littoralis NPV as ORF21), and the conserved baculovirus core genes polyhedrin, DNApol, pif-2 and lef-8 confirmed that this was indeed the case and that SpltNPV-Pak-BNG is a genuine SpltNPV variant, whereas the SpltNPV-G1 isolate we used is, in fact, a SpliNPV variant, renamed to SpliNPV-G1. The newly isolated SpltNPV-Pak-BNG has the potential for development as a biocontrol agent of S. litura in Pakistan.

White spot syndrome virus (WSSV), a rod-shaped double-stranded DNA virus, is an infectious agent causing fatal disease in shrimp farming around the globe. Within shrimp populations WSSV is transmitted very fast, however, the modes and dynamics of transmission of this virus are not well understood. In the current study the dynamics of disease transmission of WSSV were investigated in small, closed populations of Penaeus monodon and Penaeus vannamei. Pair cohabitation experiments using PCR as a readout for virus infection were used to estimate transmission parameters for WSSV in these two species. The mortality rate of contact-infected shrimp in P. monodon was higher than the rate in P. vannamei. The transmission rate parameters for WSSV were not different between the two species. The relative contribution of direct and indirect transmission rates of WSSV differed between the two species. For P. vannamei the direct contact transmission rate of WSSV was significantly lower than the indirect environmental transmission rate, but for P. monodon, the opposite was found. The reproduction ratio R0 for WSSV for these two species of shrimp was estimated to be above one: 2.07 (95%CI 1.53, 2.79) for P. monodon and 1.51 (95%CI 1.12, 2.03) for P. vannamei. The difference in R0 between the two species is due to a lower host mortality and hence a longer infectious period of WSSV in P. monodon.

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.

Chikungunya virus (CHIKV) causes acute illness characterized by fever and long-lasting arthritic symptoms. The need for a safe and effective vaccine against CHIKV infections is on the rise due to on-going vector spread and increasing severity of clinical complications. Here we report the results of a comparative vaccination-challenge experiment in mice using three different vaccine candidates produced in insect cells by recombinant baculoviruses: (i) secreted (s)E1 and (ii) sE2 CHIKV glycoprotein subunits (2 μg/immunization), and (iii) CHIKV virus-like particles (VLPs) (1 μg E2 equivalent/immunization). These experiments show that vaccination with two subsequent administrations of 1 μg of Matrix M adjuvanted CHIKV VLPs completely protected AG129 mice from lethal CHIKV challenge. Vaccination with E1 and E2 subunits provided partial protection, with half of the mice surviving but with significantly lower neutralizing antibody titres as compared to the VLP vaccinated mice. This study provides evidence that even a modest neutralizing antibody response is sufficient to protect mice from CHIKV infections. Neutralization was the prominent correlate of protection. In addition, CHIKV VLPs provide a superior immune response and protection against CHIKV-induced disease in mice as compared to individual CHIKV-sE1 and -sE2 subunits.

Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the trypanosomes, which cause human African trypanosomosis (HAT) or sleeping sickness in humans and African animal trypanosomosis (AAT) or nagana in animals. Due to the lack of effective vaccines and inexpensive drugs for HAT, and the development of resistance of the trypanosomes against the available trypanocidal drugs, vector control remains the most efficient strategy for sustainable management of these diseases. Among the control methods used for tsetse flies, Sterile Insect Technique (SIT), in the frame of area-wide integrated pest management (AW-IPM), represents an effective tactic to suppress and/or eradicate tsetse flies. One constraint in implementing SIT is the mass production of target species. Tsetse flies harbor obligate bacterial symbionts and salivary gland hypertrophy virus which modulate the fecundity of the infected flies. In support of the future expansion of the SIT for tsetse fly control, the Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture implemented a six year Coordinated Research Project (CRP) entitled “Improving SIT for Tsetse Flies through Research on their Symbionts and Pathogens”. The consortium focused on the prevalence and the interaction between the bacterial symbionts and the virus, the development of strategies to manage virus infections in tsetse colonies, the use of entomopathogenic fungi to control tsetse flies in combination with SIT, and the development of symbiont-based strategies to control tsetse flies and trypanosomosis. The results of the CRP and the solutions envisaged to alleviate the constraints of the mass rearing of tsetse flies for SIT are presented in this special issue.

The Glossinavirus (Glossina pallidipes salivary gland hypertrophy virus (GpSGHV)) is a rod-shaped enveloped insect virus containing a 190,032 bp-long, circular dsDNA genome. The virus is pathogenic for the tsetse fly Glossina pallidipes and has been associated with the collapse of selected mass-reared colonies. Maintenance of productive fly colonies is critical to tsetse and trypanosomiasis eradication in sub-Saharan Africa using the Sterile Insect Technique. Proteomics, an approach to define the expressed protein complement of a genome, was used to further our understanding of the protein composition, morphology, morphogenesis and pathology of GpSGHV. Additionally, this approach provides potential targets for novel and sustainable molecular-based antiviral strategies to control viral infections in tsetse colonies. To achieve this goal, identification of key protein partners involved in virus transmission is required. In this review, we integrate the available data on GpSGHV proteomics to assess the impact of viral infections on host metabolism and to understand the contributions of such perturbations to viral pathogenesis. The relevance of the proteome findings to tsetse and trypanosomiasis management in sub-Sahara Africa is also considered.

The Agrotis segetum granulovirus (AgseGV) F protein was previously identified as the first betabaculovirus F protein with functional homology to Autographa californica nucleopolyhedrovirus (AcMNPV) GP64. In the current study, F proteins from Xestia c-nigrum granulovirus (XecnGV), Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), Choristoneura occidentalis granulovirus (ChocGV) and Plutella xylostella GV (PlxyGV) were studied for their ability to rescue the infectivity of gp64-null AcMNPV. Our results showed that most studied betabaculovirus F proteins could replace the function of AcMNPV GP64, however, their efficiencies to rescue the infectivity of gp64-null AcMNPV were substantially different. PlxyF, although fusogenic, was the only protein that failed to substitute the function of AcMNPV GP64. Further studies using Sf90p1D cell line showed that PlxyF appeared to be properly incorporated into AcMNPV virions and underwent correct post-translational cleavage and N-linked glycosylation. However, the gp64-null AcMNPV containing PlxyF could not be propagated in either Sf9 or P. xylostella cells.

Background Glossina m. morsitans is the primary vector of the Trypanosoma brucei group, one of the causative agents of African trypanosomoses. The parasites undergo metacyclogenesis, i.e. transformation into the mammalian-infective metacyclic trypomastigote (MT) parasites, in the salivary glands (SGs) of the tsetse vector. Since the MT-parasites are largely uncultivable in vitro, information on the molecular processes that facilitate metacyclogenesis is scanty. Methods To bridge this knowledge gap, we employed tandem mass spectrometry to investigate protein expression modulations in parasitized (T. b. brucei-infected) and unparasitized SGs of G. m. morsitans. We annotated the identified proteins into gene ontologies and mapped the up- and downregulated proteins within protein-protein interaction (PPI) networks. Results We identified 361 host proteins, of which 76.6 % (n = 276) and 22.3 % (n = 81) were up- and downregulated, respectively, in parasitized SGs compared to unparasitized SGs. Whilst 32 proteins were significantly upregulated (> 10-fold), only salivary secreted adenosine was significantly downregulated. Amongst the significantly upregulated proteins, there were proteins associated with blood feeding, immunity, cellular proliferation, homeostasis, cytoskeletal traffic and regulation of protein turnover. The significantly upregulated proteins formed major hubs in the PPI network including key regulators of the Ras/MAPK and Ca2+/cAMP signaling pathways, ubiquitin-proteasome system and mitochondrial respiratory chain. Moreover, we identified 158 trypanosome-specific proteins, notable of which were proteins in the families of the GPI-anchored surface glycoproteins, kinetoplastid calpains, peroxiredoxins, retrotransposon host spot multigene and molecular chaperones. Whilst immune-related trypanosome proteins were over-represented, membrane transporters and proteins involved in translation repression (e.g. ribosomal proteins) were under-represented, potentially reminiscent of the growth-arrested MT-parasites. Conclusions Our data implicate the significantly upregulated proteins as manipulators of diverse cellular processes in response to T. b. brucei infection, potentially to prepare the MT-parasites for invasion and evasion of the mammalian host immune defences. We discuss potential strategies to exploit our findings in enhancement of trypanosome refractoriness or reduce the vector competence of the tsetse vector. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1714-z) contains supplementary material, which is available to authorized users.

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) is a dsDNA virus exclusively pathogenic to tsetse flies (Diptera; Glossinidae). The 190 kb GpSGHV genome contains 160 open reading frames and encodes more than 60 confirmed proteins. The asymptomatic GpSGHV infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (SGH). Flies with SGH show reduced general fitness and reproductive dysfunction. Although the occurrence of SGH is an exception rather than the rule, G. pallidipes is thought to be the most susceptible to expression of overt SGH symptoms compared to other Glossina species that are largely asymptomatic. Although Glossina salivary glands (SGs) play an essential role in GpSGHV transmission, the functions of the salivary components during the virus infection are poorly understood. In this study, we used mass spectrometry to study SG proteomes of G. pallidipes and G. m. morsitans, two Glossina model species that exhibit differential GpSGHV pathologies (high and low incidence of SGH, respectively). A total of 540 host proteins were identified, of which 23 and 9 proteins were significantly up- and down-regulated, respectively, in G. pallidipes compared to G. m. morsitans. Whereas 58 GpSGHV proteins were detected in G. pallidipes F1 progenies, only 5 viral proteins were detected in G. m. morsitans. Unlike in G. pallidipes, qPCR assay did not show any significant increase in virus titers in G. m. morsitans F1 progenies, confirming that G. m. morsitans is less susceptible to GpSGHV infection and replication compared to G. pallidipes. Based on our results, we speculate that in the case of G. pallidipes, GpSGHV employs a repertoire of host intracellular signaling pathways for successful infection. In the case of G. m. morsitans, antiviral responses appeared to be dominant. These results are useful for designing additional tools to investigate the Glossina-GpSGHV interactions.

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.

Sequence comparisons of the genomes of white spot syndrome virus (WSSV) strains have identified regions containing variable-length insertions/deletions (i.e. indels). Indel-I and Indel-II, positioned between open reading frames (ORFs) 14/15 and 23/24, respectively, are the largest and the most variable. Here we examined the nature of these 2 indel regions in 313 WSSV-infected Penaeus monodon shrimp collected between 2006 and 2009 from 76 aquaculture ponds in the Mekong Delta region of Vietnam. In the Indel-I region, 2 WSSV genotypes with deletions of either 5950 or 6031 bp in length compared with that of a reference strain from Thailand (WSSV-TH-96-II) were detected. In the Indel-II region, 4 WSSV genotypes with deletions of 8539, 10970, 11049 or 11866 bp in length compared with that of a reference strain from Taiwan (WSSV-TW) were detected, and the 8539 and 10970 bp genotypes predominated. Indel-II variants with longer deletions were found to correlate statistically with WSSV-diseased shrimp originating from more intensive farming systems. Like Indel-I lengths, Indel-II lengths also varied based on the Mekong Delta province from which farmed shrimp were collected.

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.

INCE, Ikbal Agah/0000-0003-2299-9946; Kariithi, Henry/0000-0002-9250-9108; ABD-ALLA, Adly/0000-0001-7540-4462 WOS: 000285221000020 PubMed: 20719992 Many species of tsetse flies (Diptera Glossinidae) can be infected by a virus that causes salivary gland hypertrophy (SGH) The genomes of viruses isolated from Glossina pallidipes (GpSGHV) and Musca domestica (MdSGHV) have recently been sequenced Tsetse flies with SGH have reduced fecundity and fertility which cause a serious problem for mass rearing in the frame of sterile insect technique (SIT) programmes to control and eradicate tsetse populations in the wild A potential intervention strategy to mitigate viral infections in fly colonies is neutralizing of the GpSGHV infection with specific antibodies against virion proteins Two major GpSGHV virion proteins of about 130 and 50 kDa, respectively, were identified by Western analysis using a polyclonal rabbit antibody raised against whole GpSHGV virions The proteome of GpSGHV, containing the antigens responsible for the immune-response, was investigated by liquid chromatography tandem mass spectrometry and 61 virion proteins were identified by comparison with the genome sequence Specific antibodies were produced in rabbits against seven candidate proteins including the ORF10/C-terminal fragment ORF47 and ORF96 as well as proteins involved in peroral infectivity PIF-1 (ORF102), PIF-2 (ORF53), PIF-3 (ORF76) and P74 (ORF1) Antiserum against ORF10 specifically reacted to the 130 kDa protein in a Western blot analysis and to the envelope protein of GpSGHV, detected by using immunogold-electron microscopy This result suggests that immune intervention of viral infections in colonies of G pallidipes is a realistic option Wageningen University and Research Centre, The NetherlandsNetherlands Government The authors would like to thank Wageningen University and Research Centre, The Netherlands for awarding a Master of Science grant to Mr Henry M Karnthi to carry out these studies We are indebted to the Entomology Unit FAO/IAEA Agriculture and Biotechnology Laboratories Seibersdorf Austria for providing the virus used in this research All proteomic LC MS/MS measurements were done at Biqualys Advanced Analysis Company, the Netherlands (www biqualys nl) We would like to thank Marc Vreysen for reviewing the manuscript

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.

The ‘independent action hypothesis’ (IAH) states that each pathogen individual has a non-zero probability of causing host death and that pathogen individuals act independently. IAH has not been rigorously tested. In this paper, we (i) develop a probabilistic framework for testing IAH and (ii) demonstrate that, in two out of the six virus–insect pathosystems tested, IAH is supported by the data. We first show that IAH inextricably links host survivorship to the number of infecting pathogen individuals, and develop a model to predict the frequency of single- and dual-genotype infections when a host is challenged with a mixture of two genotypes. Model predictions were tested using genetically marked, near-identical baculovirus genotypes, and insect larvae from three host species differing in susceptibility. Observations in early-instar larvae of two susceptible host species support IAH, but observations in late-instar larvae of susceptible host species and larvae of a less susceptible host species were not in agreement with IAH. Hence the model is experimentally supported only in pathosystems in which the host is highly susceptible. We provide, to our knowledge, the first qualitative experimental evidence that, in such pathosystems, the action of a single virion is sufficient to cause disease.

The genome of Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) contains two open reading frames, Cc-phrl and Cc-phr2, which encode putative class 11 CPD-DNA photolyases. CPD-photolyases repair UV-induced pyrimidine cyclobutane dimers using visible light as an energy source. Expression of Cc-phr2 provided photolyase deficient Escherichia coli cells with photoreactivating activity indicating that Cc-phr2 encodes an active photolyase, in contrast, Cc-phrl did not rescue the photolyase deficiency. Cc-phr2 was overexpressed in E. coli and the resulting photolyase was purified till apparent homogeneity. Spectral measurements indicated the presence of FAD, but a second chromophore appeared to be absent. Recombinant Cc-phr2 photolyase was found to bind specifically FO (8-hydroxy7,8-didemethyl-5-deazariboflavin), which is an antenna chromophore present in various photolyases.. After reconstitution, FAD and FO were present in approximately equimolar amounts. In reconstituted photolyase the FO chromophore is functionally active as judged from the increase in the in vitro repair activity. This study demonstrates for the first time that a functional photolyase is encoded by an insect virus, which may have implications for the design of a new generation of baculoviruses with improved performance in insect pest control. (c) 2008 Elsevier B.V. All rights reserved.

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.

For people with insulin-dependent diabetes mellitus (IDDM) renal disease represents a life-threatening and costly complication. The EURODIAB IDDM Complications Study, a cross-sectional, clinic-based study, was designed to determine the prevalence of renal complications and putative risk factors in stratified samples of European individuals with IDDM. The present study examined the relationship between dietary protein intake and urinary albumin excretion rate (AER). Food intake was assessed centrally by a standardized 3-day dietary record. Urinary AER was determined in a central laboratory from a timed 24-h urine collection. Complete data were available from 2696 persons with IDDM from 30 centres in 16 European countries. In individuals who reported protein consumption less than 20 % of total food energy intake, mean AER was below 20 μg/min. In those in whom protein intake constituted more than 20 %, mean AER increased, a trend particularly pronounced in individuals with hypertension and/or poor metabolic control. Trends reached statistical significance for intakes of total protein (% of energy, p = 0.01) and animal protein (% of energy, p = 0.02), while no association was seen for vegetable protein (p = 0.83). These findings support the current recommendation for people with diabetes not to exceed a protein intake of 20 % of total energy. Monitoring and adjustment of dietary protein appears particularly desirable for individuals with AER exceeding 20 μg/min (approximately 30 mg/24 h), especially when arterial pressure is raised and/or diabetic control is poor. [Diabetologia (1997) 40: 1219–1226]

Objectives: Repeatability of a dietary method is important in determining the quality of nutritional data. It should be assessed in the population of interest. This study evaluated the repeatability of nutritional data from standardized three-day dietary records, from the clinic-based, cross-sectional multi-centre EURODIAB IDDM Complications Study. Design and Subjects: 15% of the total EURODIAB cohort was randomly selected to test the repeatability of nutritional intake data. Two three-day records, completed three weeks apart, were available for 216 diabetic patients (7.5%) representative of the total cohort. All records were analysed centrally, for intakes of protein (animal and vegetable), fat (saturated fat and cholesterol), carbohydrate, fibre, alcohol and energy. Repeatability was measured comparing mean intakes, determining the proportion of patients classified into the same/opposite quartile by the two three-day records and assessing mean differences with standard deviations (s.d.d). Results: There were no significant differences in mean energy and nutrient intakes between the first and second records. Classification of individuals into the opposite quartile occurred only in 0–4% of patients and overall about 50% (range 44–74%) of the subjects were classified into the same quartiles of intakes. Only small mean differences were found for energy intake (−156 (1633) kJ; 95% confidence limits −375, 63 kJ) and nutrients with s.d.ds comparable to intra-individual variations in the general population. The differences in energy intake were randomly distributed over the range of intakes. Conclusions: The present study demonstrates that standardized three day dietary records show a high degree of repeatability within a short period of time in a sample of European IDDM patients. The good repeatability strengthens the conclusions drawn from the nutritional data in the EURODIAB IDDM Complications Study. Sponsorship: Nutrition Co-ordinating Centre research funds, Diabetes Research Institute at Heinrich-Heine University, Dusseldorf. The EURODIAB IDDM Complications Study was supported by the European Community.

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 delayed-early DNA polymerase promoter of Chilo iridescent virus (CIV), officially known as Invertebrate iridescent virus, was fine mapped by constructing a series of increasing deletions and by introducing point mutations. The effects of these mutations were examined in a luciferase reporter gene system using Bombyx mori cells transfected with promoter constructs and infected with CIV. When the size of the upstream element was reduced from position −19 to −15, relative to the transcriptional start site, the luciferase activity was reduced to almost zero. Point mutations showed that each of the 5 nt (AAAAT) located between –19 and –15 were equally essential for promoter activity. Mutations at individual bases around the transcription initiation site showed that the promoter extended until position −2 upstream of the transcription start site. South-Western analysis showed that a protein of approximately 100 kDa interacted with the −19 nt promoter fragment in CIV-infected cells. This binding did not occur with a point mutant that lacked promoter activity. The AAAAT motif was also found in the DNA polymerase promoter region of other iridoviruses and in other putative CIV delayed-early genes.

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

It is both a great pleasure and an honour for me to give the annual Founders’ Lecture of the Society for Invertebrate Pathology. That the 2006 meeting is being held in Wuhan, China, is a golden opportunity to honor a man who made a seminal contribution to the field of invertebrate pathology and who was a pioneer in China, Professor Shang yin Gao, also known as Gaw Shan yin in the older literature (Fig. 1). I compliment the Founders’ Lecture committee for their insight in honoring this distinguished Chinese scientist at our meeting in Wuhan. It is also very appropriate to address you today since we celebrate the 50th anniversary of the foundation of the Wuhan Institute of Virology by Professor Gao. And finally, it is a personal pleasure to look back at my personal connection with Professor Gao and his legacy, which culminated in intensive and fruitful collaborations for almost 15 years with the Institute he founded. I had the good fortune to meet Professor Gao personally on two occasions. The first time was in Prague in 1978, when Professor Gao was invited to attend the SIP meeting. We did not get a chance to talk, but this was compensated a year later when he attended the SIP meeting in Gainesville, FL, from where we travelled together to College Station, TX, to visit Dr. Max Summers, who is now known for his studies on baculoviruses and polydnaviruses. We flew in a small airplane and I was seated next to Professor Gao. However, one seat did not fit his size and I changed one seat over and a lively discussion followed. He was very interested in what I was doing, in particular the molecular methods that I used at that time to identify baculoviruses using restriction enzymes. He invited me to come to China which, however, did not materialize until after his death. I vividly remember his openness and frankness, quite

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.

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.

White spot syndrome virus (WSSV) is a major disease in crustaceans, particularly shrimp, due to the current intensity of aquaculture practices. Novel strategies including vaccination to control this virus would be highly desirable. However, invertebrates lack a true adaptive immune response system and seem to rely on various innate immune responses. An alternative and more specific approach to counteract WSSV infections in shrimp could be by the exploitation of RNA interference. As long dsRNA molecules induce a general, sequence-independent anti-viral immunity in shrimp [Robalino, J., Browdy, C.L., Prior, S., Metz, A., Parnell, P., Gross, P., Warr, G., 2004. J. Virol. 78, 10442-10448], it was investigated whether shorter 21 nt siRNAs with homology to the WSSV vp15 and vp28 genes would give a sequence-specific interference response in the shrimp Penaeus monodon. Vp28 siRNAs as well as nonspecific control gfp siRNAs were able to specifically and efficiently silence their homologous genes in a heterologous baculovirus insect cell expression system. However, in shrimps no such a specific effect was observed. Shrimp injected with vp15 or vp28 siRNAs before WSSV challenge gave a significantly lower mortality rate, but not significantly different when shrimps were injected with gfp siRNA. Thus, large dsRNA molecules as well as siRNAs induce a sequence-independent anti-viral immunity when injected in shrimp.

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.

We report on the yield of Polyhedral Inclusion Bodies (PIBs) from first to fifth instar Helicoverpa armigera larvae inoculated with wild-type Helicoverpa armigera nucleopolyhedrovirus (HaSNPV-WT) or with one of two HaSNPV recombinants, one in which the ecdysteroid UDP-glucosyltransferase (egt) gene was deleted (HaSNPV-EGTD) and a second in which the egt gene was replaced by a scorpion toxin (AaIT) gene (HaSNPV-AaIT). A significant linear relationship between the logarithm of cadaver weight and the logarithm of the number of PIBs per cadaver was observed for all three virus types. The increase of the number of PIBs with larval weight was significantly greater for HaSNPV-WT than for the recombinant viruses. For each of the three HaSNPVs, PIB yield per cadaver was significantly affected by larval instar at death and by time to death, with later instars and longer surviving larvae producing a greater number of PIBs. As both recombinants caused host larvae to die at earlier instars than HaSNPV-WT, their virus yields were significantly reduced. Virus yield per larva, inoculated with HaSNPV-AaIT in the first, second, third, fourth or fifth larval stage was 23, 32, 41, 44 and 47% of the yield of HaSNPV-WT, respectively. For HaSNPV-EGTD, virus yield per larva inoculated in first through fifth instar, respectively, was 41, 55, 63, 54 and 82% of the yield of HaSNPV-WT. These results provide a basis for optimizing the production regime of recombinant HaSNPVs in larvae and for modeling the behavior of these viruses in agro-ecosystems

The satin moth Leucoma salicis L. (Lepidoptera, Lymantriidae) is a frequent defoliator of poplar trees (Populus spp.) in Europe and Asia (China, Japan). Around 1920 the insect was introduced into the USA and Canada. In this paper, a multicapsid nucleopolyhedrovirus isolated from L. salicis larvae in Poland (LesaNPV) was characterized and appeared to be a variant of Orgyia pseudotsugata (Op) MNPV. O. pseudotsugata, the Douglas fir tussock moth (Lepidoptera, Lymantriidae), occurs exclusively in North America. Sequences of three conserved baculovirus genes, polyhedrin, lef-8, and pif-2, were amplified in polymerase chain reactions using degenerate primer sets, and revealed a high degree of homology to OpMNPV. Restriction enzyme analysis confirmed the close relationship between LesaNPV and OpMNPV, although a number of restriction fragment length polymorphisms were observed. The lef-7 gene, encoding late expression factor 7, and the ctl-2 gene, encoding a conotoxin-like protein, were chosen as putative molecular determinants of the respective viruses. The ctl-2 region appeared suitable for unequivocal identification of either virus as LesaNPV lacked a dUTPase gene in this region. Our observations may suggest that LesaNPV, along with L. salicis, was introduced into O. pseudotsugata after introduction of the former insect into North America in the 1920s.