Baculovirus Per Os Infectivity Factors Form a Complex on the Surface of Occlusion-Derived Virus

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% ß-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 infectio

Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells

East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five™ cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP[ratio]p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF

Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective conditio

Proteomic analysis of Glossina pallidipes salivary gland hypertrophy virus virions for immune intervention in tsetse fly colonies

Many species of tsetse flies (Diptera: Glossinidae) can be infected by a virus that causes salivary gland hypertrophy (SGH). The viruses isolated from Glossina pallidipes (GpSGHV) and Musca somestica (MdSGHV) have recently been sequenced. Tsetse flies with SGH have a reduced fecundity and fertility which cause a serious problem for mass rearing in the frame of sterile insect technique (SIT) programs 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 kDa and 50 kDa, respectively, were identified by Western analysis using 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 (LC-MS/MS) 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 of GpSGHV using immunogold-EM. This result suggests that immune intervention of viral infections in colonies of G. pallidipes is a realistic optio

Effectiveness of alcohol prevention interventions based on the principles of social marketing:A systematic review

Background Alcohol education aims to increase knowledge on the harm related to alcohol, and to change attitudes and drinking behaviour. However, little (lasting) evidence has been found for alcohol education, in changing alcohol-related attitudes and behaviour. Social marketing uses marketing techniques to achieve a social or healthy goal, and can be used in alcohol education. Social marketing consists of eight principles: customer orientation, insight, segmentation, behavioural goals, exchange, competition, methods mix, and is theory based. This review investigates the application of social marketing in alcohol prevention interventions, and whether application of social marketing influences alcohol-related attitudes or behaviour. Method A literature search was conducted in PubMed, PsychInfo, Cochrane and Scopus. Inclusion criteria were that original papers had to describe the effects of an alcohol prevention intervention developed according to one or more principles of social marketing. No limits were set on the age of the participants or on the kind of alcohol prevention intervention. The abstracts of the 274 retrieved studies were reviewed and the full texts of potentially relevant studies were screened. Results Six studies met the inclusion criteria and were included in this review. These six studies showed associations for the application of social marketing techniques on alcohol-related attitudes or behaviour; one study relates to participation in a drinking event, four to alcohol drinking behaviour, two to driving a car while under the influence of alcohol, two to recognition of campaign messages or campaign logo, and one to awareness of the campaign. However, no associations were also found. In addition, the studies had several limitations related to a control group, response rate and study methodology. Conclusion Based on this review, the effect of applying the principles of social marketing in alcohol prevention in changing alcohol-related attitudes or behaviour could not be assessed. More research, with a good quality methodology, like using a randomized control trial and measuring short, medium, and long-term effects, is required on this topic. Policy implications are discussed. Keywords: Alcohol, Prevention, Effectiveness, Social marketing, Interventio

Een nieuw baculovirus van en voor de Turkse mot

Samenvatting van de voordracht te houden op 30 november 2005 tijdens de Najaarsvergadering van de KNPV (Koninklijke Nederlandse Plantenziektekundige Vereniging). Onderzoek naar de mogelijke toepasbaarheid van een baculovirus uit de Turkse mot voor biologische bestrijding van de rups van deze mo

Essential C-Terminal region of the baculovirus minor capsid protein VP80 binds DNA

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

Edible insects unlikely to contribute to transmission of coronavirus SARS-CoV-2

In the context of food safety, edible insects are evaluated for biological hazards such as microbial pathogens according to regulations currently in place. When the European Food Safety Authority evaluated the hazards of edible insects as a potential source of pathogenic viruses for humans and livestock, the novel zoonotic coronavirus SARS-CoV-2 had not yet emerged but other pathogenic coronaviruses such as SARS (SARS-CoV) and MERS (MERS-CoV) were known. As a result of the COVID-19 pandemic, animal sources of protein for human consumption are being evaluated for the risks of being a transmission vector of coronaviruses, like SARS-CoV-2. Insects lack a receptor that can bind SARS-CoV-2, thus preventing the virus from replicating in insects, unlike some vertebrate livestock species and companion animals. Despite extensive monitoring, coronaviruses have never been recorded in insect microbiomes. Contamination of insects produced for food or feed may occur during the production process, resulting from rearing substrate or from insect farmers. However, the currently permitted rearing substrates do not include animal products and the farming process is highly automated, thus limiting interactions between farmers and insects. If contamination would still occur, the fact that the insects in production are not hosts to SARS-CoV-2 precludes virus replication and the further processing of the insects will destroy the contamination. We conclude that the hazard of edible insects being a transmission vector of SARS-CoV-2 is extremely low.</p

Characterization of novel components of the baculovirus per os infectivity factor complex

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