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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.

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.

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.

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.

The genome organization of the Helicoverpa armigera single-nucleo-capsid nucleopolyhedrovirus (HearNPV) was investigated and compared to that of other baculoviruses. A detailed physical map was constructed for the restriction enzymes BamHI, BglII, EcoRI, HindIII, KpnI, PstI, SacI and XhoI. The 130.1-kilobase pairs (kb) long viral genome was cloned as restriction fragments into a plasmid library, which covered 97.5% of the viral genome. From this plasmid library about 45 kb of dispersed sequence information was generated. Fifty-three putative open reading frames (ORFs) with homology to ORFs of other baculoviruses were identified and their location on the genome of HearNPV was determined. The arrangement of the ORFs along the HearNPV genome is significantly different from the multiple-nucleocapsid NPVs, AcMNPV, BmNPV and OpMNPV, which have a highly collinear genome, and the granulovirus XcGV. In contrast the genomes of HearNPV and SeMNPV are highly collinear. This close relatedness between an MNPV and an SNPV is supported by the phylogeny of specific genes of these two viruses and suggests that the morphotype (S or M) is only a taxonomic and not a phylogenetic denominator. The data presented here also give further support to the view that the gene distribution along baculovirus genomes can be used as a character to describe baculovirus relatedness independent of gene phylogeny. Five homologous regions (hrs) were located on the genome of HearNPV. This is also the first report of the existence of hrs in SNPVs and this observation suggests that hrs are a common feature of baculovirus genomes.

The genomic position and nucleotide sequence of the immediate early gene ie1 of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) were determined. The SeMNPV ie1 gene had the potential to encode a protein of 714 amino acids with a predicted molecular mass of 82.0 kDa, representing the largest baculovirus IE1 known to date. The similarity of SeMNPV IE1 with IE1 proteins from other baculoviruses was restricted to the basic C-terminal two-thirds of the protein, which is involved in DNA binding. A single ie1 transcript of 2.5 kb was detected at 1 h p.i., peaking at 8 h p.i. and disappearing at 24 h p.i. From 8 h p.i. onwards a 1.7 kb transcript was detected, which became more abundant late in infection. Primer extension analysis revealed the use of several start sites early and late in infection. One of these was located in the promoter motif CAGT located at position -15 relative to the translational start codon, most intensively used at 8 h p.i. Four kb upstream of the SeMNPV ie1 gene, ORF xd244 was identified with homology to the used left ie0 exon of AcMNPV, OpMNPV and LdMNPV. Phylogenetic analysis indicated that the SeMNPV IE1 protein shared the most recent ancestor with its HzSNPV and LdMNPV homologues, although their IE1 proteins had diverged considerably from each other and from other baculoviruses.

By injection of a liposomal complex containing baculovirus DNA directly into the hemocoel of insect larvae a polyhedrosis disease was induced. After cotransfection of insect larvae with circular viral DNA and transfer vector DNA recombinant viruses were generated with a frequency of about 2%, similar to what is obtained in vitro using insect cell cultures. Based on these results an alternative strategy for the generation of recombinants can be derived for baculoviruses for which susceptible cell lines are not readily available. This strategy involves the injection of baculovirus DNA into susceptible larvae followed by in vivo cloning.

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons.

An ecdysteroid UDP-glucosyltransferase (egt) gene was identified from the single (S) nucleocapsid nucleopolyhedrovirus of Heliothis armigera (HearNPV). In baculovirus-infected insects the viral enzyme (EGT) plays a pivotal role in abrogating the insect molting process. The open reading frame of the egt gene is 1545 nucleotides long, encoding a putative protein of 515 amino acids with a Mr of 59.1. The 5'-noncoding region contains a putative early (CAGT) and late (TAAG) motif for transcription initiation, a transcription enhancer sequence (CGTCGC) and two TATA boxes. A putative polyA signal, AATAAA, was found downstream of the translation stop codon. A putative signal peptide of 21 residues was present at the N-terminus of the EGT. The HearNPV egt gene has a high degree of nucleotide and amino acid sequence homology to the egt genes of Buzura suppressaria SNPV and Spodoptera exigua MNPV. The HearNPV EGT shares ten conserved motifs with other EGTs. A phylogenetic tree of twelve baculovirus EGTs was constructed by using maximum parsimony analysis, suggesting that SNPVs do not form a separate clade within the baculovirus family.

In this chapter we have attempted to evaluate the most important parameters which can be useful for the pur-pose of design and scale up. Insect cells and animal cells in general can be grown well in large vessels. However, none of the theories and parameters discussed in this chapter have been validated on a larger scale than laboratory and small pilot reactors. Selection of the most suitable design and scale-up method there-fore needs in particular studies in larger vessels. The Kolmogorov theory and the killing-volume model are in this respect the most promising approaches for the optimal design of large-scale animal-cell bioreactors.

Cauliflower mosaic virus (CaMV) gene I encodes a protein (P1) that has been implicated in the control of virus movement in infected plants. To assist in the characterization of the mechanism of action of P1, gene I has been expressed efficiently in Spodoptera frugiperda (Sf) cells using recombinant baculovirus. Control of the expression of CaMV gene I by the polyhedrin late promoter in the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) resulted in very high levels of P1 accumulation late in the infection cycle. This was predominantly as insoluble inclusion bodies within the cytoplasm of infected Sf cells, and not extracellularly. Evidence from anomalous gel migration and sequence homology with an analogous viral protein (tobacco mosaic virus 30K) indicated that P1 may be post-translationally processed. However, neither phosphorylation nor glycosylation of P1 occurred in this system, suggesting a functional distinction between P1 and TMV 30K. P1 from insect cells and native P1 from infected plants were immunologically related, allowing the expressed product to be used in the preparation of anti-P1 serum for detecting P1 in plant extracts. The full-size (46 kD) P1 product from insect cells, from plants, and from in vitro translations of in vitro gene I transcripts all showed similar behavior on two-dimensional protein gels, with a major pI of 7.0. Using a combination of 4 M urea, 1 M NaCl, and high temperature, P1 was solubilized. Approximately 5% of the starting material remained in solution after dialysis and remained stable to freeze/thawing. This preparation should enable us to identify the biochemical function of P1 and to resolve its role in controlling virus spread.

A mathematical model for the design of bubble-columns for growth of shear-sensitive insect cells is presented. The model is based on two assumptions. First, the loss of cell viability as a result of aeration is a first-order process. Second, a hypothetical volume X, in which all viable cells are killed, is associated with each air bubble during its lifetime. The model merely consists of an equation for k d, the first-order death-rate constant, and A min, the minimum specific surface area of the air bubbles to supply sufficient oxygen. In addition to X, the equation for k d contains the air flow F, the air-bubble diameter d b, the diameter D and the height H of the bubble column. This equation has been experimentally validated. Comparison of the equations for k d and A min shows that especially H is the key parameter to manipulate in bubble-column design in order to meet the demands set by A min and k d

A model, suitable for reactor-design purposes, is presented for the infection process of insect cells with baculovirus in a continuous bioreactor system. The infection process can be described by a first-order reaction mechanism if the viable, non-infected, insect cells are regarded as substrate, and the resulting polyhedra as product. Experimental results from continuous systems, consisting of a series of mixed reactors in which growth of Spodoptera frugiperda cells takes place in the first reactor and infection with Autographa californica nuclear polyhedrosis virus in the other reactors in the series, show typical values for the reaction rate constant of 0.7–1.0 10-5 s-1.