Replication-independent change in the frequencies of distinct genome segments of a multipartite virus during its transit within aphid vectors.

Multipartite viruses exhibit a fragmented genome composed of several nucleic acid segments individually packaged in distinct viral particles. The genome of all species of the genus Nanovirus holds eight segments, which accumulate at a very specific and reproducible relative frequency in the host plant tissues. In a given host species, the steady state pattern of the segments' relative frequencies is designated the genome formula and is thought to have an adaptive function through the modulation of gene expression. Nanoviruses are aphid-transmitted circulative non-propagative viruses, meaning that the virus particles are internalized into the midgut cells, transferred to the hemolymph, and then to the saliva, with no replication during this transit. Unexpectedly, a previous study on the faba bean necrotic stunt virus revealed that the genome formula changes after ingestion by aphids. We investigate here the possible mechanism inducing this change by first comparing the relative segment frequencies in different compartments of the aphid. We show that changes occur both in the midgut lumen and in the secreted saliva but not in the gut, salivary gland, or hemolymph. We further establish that the viral particles differentially resist physicochemical variations, in particular pH, ionic strength, and/or type of salt, depending on the encapsidated segment. We thus propose that the replication-independent genome formula changes within aphids are not adaptive, contrary to changes occurring in plants, and most likely reflect a fortuitous differential degradation of virus particles containing distinct segments when passing into extra-cellular media such as gastric fluid or saliva. IMPORTANCE: The genome of multipartite viruses is composed of several segments individually packaged into distinct viral particles. Each segment accumulates at a specific frequency that depends on the host plant species and regulates gene expression. Intriguingly, the relative frequencies of the genome segments also change when the octopartite faba bean necrotic stunt virus (FBNSV) is ingested by aphid vectors, despite the present view that this virus travels through the aphid gut and salivary glands without replicating. By monitoring the genomic composition of FBNSV populations during the transit in aphids, we demonstrate here that the changes take place extracellularly in the gut lumen and in the saliva. We further show that physicochemical factors induce differential degradation of viral particles depending on the encapsidated segment. We propose that the replication-independent changes within the insect vector are not adaptive and result from the differential stability of virus particles containing distinct segments according to environmental parameters.

Extended in vivo transcriptomes of two ascoviruses with different tissue tropisms reveal alternative mechanisms for enhancing virus reproduction in hemolymph.

Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.

Global Metabolic Profiling of Baculovirus Infection in Silkworm Hemolymph Shows the Importance of Amino-Acid Metabolism.

Viruses rely on host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Infection of the silkworm, Bombyx mori, by Bombyx mori nucleopolyhedrovirus (BmNPV), has been studied extensively in the past to unravel interactions between baculoviruses and their lepidopteran hosts. To understand the interaction between the host metabolic responses and BmNPV infection, we analyzed global metabolic changes associated with BmNPV infection in silkworm hemolymph. Our metabolic profiling data suggests that amino acid metabolism is strikingly altered during a time course of BmNPV infection. Amino acid consumption is increased during BmNPV infection at 24 h post infection (hpi), but their abundance recovered at 72 hpi. Central carbon metabolism, on the other hand, particularly glycolysis and glutaminolysis, did not show obvious changes during BmNPV infection. Pharmacologically inhibiting the glycolytic pathway and glutaminolysis also failed to reduce BmNPV replication, revealing that glycolysis and glutaminolysis are not essential during BmNPV infection. This study reveals a unique amino acid utilization process that is implemented during BmNPV infection. Our metabolomic analysis of BmNPV-infected silkworm provides insights as to how baculoviruses induce alterations in host metabolism during systemic infection.

Transmission parameters of pepper whitefly-borne vein yellows virus (PeWBVYV) by Bemisia tabaci and identification of an insect protein with a putative role in polerovirus transmission.

Pepper crops in Israel are infected by poleroviruses, Pepper vein yellows virus 2 (PeVYV-2) and Pepper whitefly-borne vein yellows virus (PeWBVYV). Herein we characterize the transmission of PeWBVYV and the aphid-transmitted PeVYV-2, and show that PeWBVYV is specifically transmitted by MEAM1 species of the whitefly Bemisia tabaci, with a minimum latency period of 120 h, and not by the Mediterranean (MED). PeWBVYV and PeVYV-2 were detected in the hemolymph of MED and MEAM1, respectively, however, amounts of PeWBVYV in the hemolymph of MED or PeVYV-2 in MEAM1 were much lower than PeWBVYV in hemolymph of MEAM1. Moreover, we show that PeWBVYV does not interact with the GroEL protein of the symbiont Hamiltonella and thus does not account for the non-transmissibility by MED. An insect glycoprotein, C1QBP, interacting in vitro with the capsid proteins of both PeWBVYV and PeVYV-2 is reported which suggests a putative functional role in polerovirus transmission.

A Tale of Two Transcriptomic Responses in Agricultural Pests via Host Defenses and Viral Replication.

Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus that causes systemic infections in many arthropod pests. The specific molecular processes underlying the biocidal activity of AcMNPV on its insect hosts are largely unknown. We describe the transcriptional responses in two major pests, Spodoptera frugiperda (fall armyworm) and Trichoplusia ni (cabbage looper), to determine the host-pathogen responses during systemic infection, concurrently with the viral response to the host. We assembled species-specific transcriptomes of the hemolymph to identify host transcriptional responses during systemic infection and assessed the viral transcript abundance in infected hemolymph from both species. We found transcriptional suppression of chitin metabolism and tracheal development in infected hosts. Synergistic transcriptional support was observed to suggest suppression of immune responses and induction of oxidative stress indicating disease progression in the host. The entire AcMNPV core genome was expressed in the infected host hemolymph with a proportional high abundance detected for viral transcripts associated with replication, structure, and movement. Interestingly, several of the host genes that were targeted by AcMNPV as revealed by our study are also targets of chemical insecticides currently used commercially to control arthropod pests. Our results reveal an extensive overlap between biological processes represented by transcriptional responses in both hosts, as well as convergence on highly abundant viral genes expressed in the two hosts, providing an overview of the host-pathogen transcriptomic landscape during systemic infection.

Comparative response of Spodoptera litura challenged per os with Serratia marcescens strains differing in virulence.

Host plays an important role in influencing virulence of a pathogen and efficacy of a biopesticide. The present study was aimed to characterize the possible factors present in Spodoptera litura that influenced pathogenecity of orally ingested S. marcescens strains, differing in their virulence. Fifth instar larvae of S. litura responded differently as challenged by two Serratia marcescens strains, SEN (virulent strain, LC50 7.02 103 cfu/ml) and ICC-4 (non-virulent strain, LC50 1.19 1012 cfu/ml). Considerable increase in activity of lytic enzymes protease and phospholipase was recorded in the gut and hemolymph of larvae fed on diet supplemented with S. marcescens strain ICC-4 as compared to the larvae treated with S. marcescens strain SEN. However, a significant up-regulation of antioxidative enzymes SOD (in foregut and midgut), CAT (in the midgut) and GST (in the foregut and hemolymph) was recorded in larvae fed on diet treated with the virulent S. marcescens strain SEN in comparison to larvae fed on diet treated with the non-virulent S. marcescens strain ICC-4. Activity of defense related enzymes lysozyme and phenoloxidase activity were also higher in the hemolymph of larvae fed with diet treated with S. marcescens strain SEN as compared to hemolymph of S. marcescens strain ICC-4 treated larvae. More number of over-expressed proteins was observed in the gut and hemolymph of S. marcescens strains ICC-4 and SEN treated larvae, respectively. Identification of the selected differentially expressed proteins indicated induction of proteins involved in insect innate immune response (Immunoglobulin I-set domain, Apolipophorin III, leucine rich repeat and Titin) in S. marcescens strain SEN treated larvae. Over-expression of two proteins, actin related protein and mt DNA helicase, were noted in S. marcescens treated larvae with very high levels observed in the non-virulent strain. Up-regulation of homeobox protein was noted only in S. marcescens strain ICC-4 challenged larvae. This study indicated that ingestion of non-virulent S. marcescens strain ICC-4 induced strong immune response in insect gut while there was weak response to the virulent S. marcescens strain SEN which probably resulted in difference in their virulence.

Metabolic responses of whiteleg shrimp to white spot syndrome virus (WSSV).

Outbreaks of white spot syndrome virus (WSSV) have caused serious damage to penaeid shrimp aquaculture worldwide. Despite great efforts to characterize the virus, the conditions that lead to infection and the infection mechanisms, there is still a lack of understanding regarding these complex virus-host interactions, which is needed to develop consistent and effective treatment methods for WSSV. In this study, we used a gas chromatography - mass spectrometry (GC-MS)-based metabolomics approach to compare the metabolite profiles of gills, haemolymph and hepatopancreas from whiteleg shrimp (Penaeus vannamei) exposed to WSSV and corresponding controls. The results revealed clear discriminations between metabolite profiles of WSSV-challenged shrimp and controlled shrimp in each tissue. The responses of shrimp gills to WSSV infection were characterized by increases of many fatty acids and amino acids in WSSV-challenged shrimp compared to the controls. Changes in haemolymph metabolite profiles include the increased levels of itaconic acid, energy-related metabolites, metabolites in glutathione cycle and decrease of amino acids. The WSSV challenge led to the decreases of several fatty acids and amino acids and increases of other amino acids, lactic acid and other organic compounds (levulinic acid, malonic acid and putrescine) in hepatopancreas. These alterations of shrimp metabolites suggest several immune responses of shrimp to WSSV in a tissue-specific manner, including upregulation of osmoregulation, antimicrobial activity, metabolic rate, gluconeogenesis, glutathione pathway in control of oxidative stress and shift from aerobic to anaerobic metabolism in shrimp which indicates the Warburg effect. The findings from this study provide a better understanding of molecular process of shrimp response against WSSV invasion which may be useful for development of disease management strategies.

The antiviral immunity of ticks against transmitted viral pathogens.

Ticks, being obligate hematophagous arthropods, are exposed to various blood-borne pathogens, including arboviruses. Consequently, their feeding behavior can readily transmit economically important viral pathogens to humans and animals. With this tightly knit vector and pathogen interaction, the replication and transmission of tick-borne viruses (TBVs) must be highly regulated by their respective tick vectors to avoid any adverse effect on the ticks' biological development and viability. Knowledge about the tick-virus interface, although gaining relevant advances in recent years, is advancing at a slower pace than the scientific developments related to mosquito-virus interactions. The unique and complicated feeding behavior of ticks, compared to that of other blood-feeding arthropods, also limits the studies that would further elaborate the antiviral immunity of ticks against TBVs. Hence, knowledge of molecular and cellular immune mechanisms at the tick-virus interface, will further elucidate the successful viral replication of TBVs in ticks and their effective transmission to human and animal hosts.

A Plant Virus Ensures Viral Stability in the Hemolymph of Vector Insects through Suppressing Prophenoloxidase Activation.

Most plant viruses require vector insects for transmission. Viral stability in the hemolymph of vector insects is a prerequisite for successful transmission of persistent plant viruses. However, knowledge of whether the proteolytic activation of prophenoloxidase (PPO) affects the stability of persistent plant viruses remains elusive. Here, we explored the interplay between rice stripe virus (RSV) and the PPO cascade of the vector small brown planthopper. Phenoloxidase (PO) activity was suppressed by RSV by approximately 60%. When the PPO cascade was activated, we found distinct melanization around RSV particles and serious damage to viral stability in the hemolymph. Viral suppression of PO activity was derived from obstruction of proteolytic cleavage of PPOs by binding of the viral nonstructural protein NS3. These results indicate that RSV attenuates the PPO response to ensure viral stability in the hemolymph of vector insects. Our research provides enlightening cues for controlling the transmission of vector-borne viruses.IMPORTANCE Large ratios of vector-borne plant viruses circulate in the hemolymph of their vector insects before entering the salivary glands to be transmitted to plants. The stability of virions in the hemolymph is vital in this process. Activation of the proteolytic prophenoloxidase (PPO) to produce active phenoloxidase (PO) is one of the major innate immune pathways in insect hemolymph. How a plant virus copes with the PPO immune reaction in its vector insect remains unclear. Here, we report that the PPO affects the stability of rice stripe virus (RSV), a notorious rice virus, in the hemolymph of a vector insect, the small brown planthopper. RSV suppresses PPO activation using viral nonstructural protein. Once the level of PO activity is elevated, RSV is melanized and eliminated from the hemolymph. Our work gives valuable clues for developing novel strategies for controlling the transmission of vector-borne plant viruses.

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR.

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

A Combination of Real-Time PCR and High-Resolution Melting Analysis to Detect and Identify CpGV Genotypes Involved in Type I Resistance.

Cydia pomonella granulovirus, in particular CpGV-M isolate, is used as a biological control against the codling moth (CM), Cydia pomonella. As a result of intensive control over the years, codling moth populations have developed resistance against this isolate. This resistance is now called type I resistance. Isolates, among them, CpGV-R5, have been found that are able to overcome type I resistance. Both CpGV-M and CpGV-R5 are used in orchards to control the codling moth. High resolution melting (HRM) has been adapted to differentiate between CpGV-M and CpGV-R5 isolates. Specific PCR primers have been designed for the CpGV p38 gene, encompassing the variable region responsible for the ability to overcome resistance. Because each amplicon has a specific melting point, it is possible to identify the CpGV-M and CpGV-R5 genotypes and to quantify their relative proportion. This method has been validated using mixtures of occlusion bodies of each isolate at various proportions. Then, the HRM has been used to estimate the proportion of each genotype in infected larvae or in occlusion bodies (OBs) extracted from dead larvae. This method allows a rapid detection of genotype replication and enables the assessment of either success or failure of the infection in field conditions.

Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp.

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies.

Resistant silkworm strain block viral infection independent of melanization.

Melanization mediated by the prophenoloxidase-activating system (proPO) is an important immune response in invertebrates. However, the role of melanization on viral infection has not been wildly revealed in Bombyx mori (B. mori), silkworm. Here, we investigated the extent of melanization of susceptible (871) and resistant (near-isogenic line 871C) B. mori strains. The result showed that the extent of melanization was significantly higher in the susceptible strain than in the resistant strain. A majority of Serpins were up-regulated in the resistant strain through iTRAQ-based quantitative proteomics, comparing with susceptible strain. Our data further identified that Serpin-5, Serpin-9 and Serpin-19 reduced PO activity, indicating that the menlanization pathway was inhibited in the resistant strain. Moreover, our results indicated that the hemolymph of 871 reduced viral infection in the presence of PTU, indicating that melanization of 871 strain hemolymph blocked viral infection. However, viral infection was significantly suppressed in the hemolymph of 871C strain regardless of the presence of PTU or not, which implied that the resistant strain inhibited viral infection independent of the melanization pathway. Taken together, our findings indicated that the melanization pathway was inhibited in resistant strain. These results expend the analysis of melanization pathway in insects and provide insights into understanding the antiviral mechanism.

Presence of Penaeus monodon densovirus in the ovary of chronically infected P. monodon subadults.

Shrimp infected with Penaeus monodon densovirus (PmoDNV) usually display no specific gross signs, but heavy infections can kill postlarvae and retard juvenile growth. In the present study, samples of hepatopancreas, feces, gonads and hemolymph were isolated from male and female P. monodon subadults chronically infected by PmoDNV. Each sample of hepatopancreas and gonad was divided into 2 parts: one for PmoDNV detection by polymerase chain reaction (PCR), and the other for routine histology and immunohistochemistry. The frequency of positive findings via PCR assays was 92% in the hepatopancreas, 57% in feces, 50% in ovary, 35% in hemolymph and 0% in the testis. Using the densitometric value (DV) of the specific band for PmoDNV relative to that of the ß-actin gene as an index of the viral load in the samples, no significant differences were observed among sample types and sexes. Hematoxylin-eosin staining of infected hepatopancreas revealed typical PmoDNV inclusions in the nuclei of infected cells. The ovaries with high DV (>1) contained various types of inclusions along the row of the follicular cells or possibly in the connective tissue cells surrounding the oocytes. Using immunohistochemistry with specific probes to detect PmoDNV proteins, a positive reaction was observed in viral inclusions found in infected hepatopancreas and in ovaries with high DV, specifically in the ovarian capsule, hemolymph, oocytes and nuclear inclusions. These results suggest that the localization of PmoDNV in P. monodon is not confined to the hepatopancreas, but rather that the virus can also occur in the ovary; hence, trans-ovarian, vertical transmission of the virus is highly possible.

Genomic analysis of a novel isolate Heliothis virescens ascovirus 3i (HvAV-3i) and identification of ascoviral repeat ORFs (aros).

Ascoviruses are circular double-stranded DNA viruses that infect insects. Herein we sequenced and analyzed the genome of the previously unrecorded ascovirus isolate Heliothis virescens ascovirus 3i (HvAV-3i). The genome size is 185,650 bp with 181 hypothetical open reading frames (ORFs). Additionally, definition based on ascovirus repeated ORFs (aros) is proposed; whereby the 29 aros from all sequenced Ascoviridae genomes are divided into six distinct groups. The topological relationship among the isolates of Heliothis virescens ascovirus 3a is (HvAV-3f, {HvAV-3h, [HvAV-3e, (HvAV-3g, HvAV-3i)]}) with every clade well supported by a Bayesian posterior probability of 1.00 and a Bootstrap value of 100%.

Differential efficiency of a begomovirus to cross the midgut of different species of whiteflies results in variation of virus transmission by the vectors.

Begomoviruses are important crop viral disease agents, and they are transmitted by whiteflies of the Bemisia tabaci complex. Although the transmission of begomoviruses by whiteflies has been studied for many years, the mechanisms governing differential transmission of begomoviruses by different species of the Bemisia tabaci complex remain largely unknown. Here we firstly compared the transmission efficiency of tobacco curly shoot virus (TbCSV) by four species of the B. tabaci complex and found that Asia II 1 transmitted this virus with the highest efficiency, whereas MEAM1 transmitted it with the lowest. Next, by performing quantitative analysis of virus and immune-fluorescence detection, we found that the efficiency of TbCSV to cross the midgut wall was higher in Asia II 1 than in MEAM1. Finally, we set the quantities of virions in the haemolymph to the same level in Asia II 1 and MEAM1 via injection and then compared their capacity in TbCSV transmission, and found that the difference in TbCSV transmission between them became smaller. Taken together, our findings suggest that the efficiency of a begomovirus to cross the midgut wall of a whitefly to reach the vector's haemolymph plays a significant role in determining transmission of the virus.

A novel white spot syndrome virus protein WSSV164 controls prophenoloxidases, PmproPOs in shrimp melanization cascade.

Melanization, mediated by the prophenoloxidase (proPO)-activating system, is an important innate immune response in invertebrates. The implication of the proPO system in antiviral response and the suppression of host proPO activation by the viral protein have previously been demonstrated in shrimp. However, the molecular mechanism of viral-host interactions in the proPO cascade remains largely unexplored. Here, we characterized the viral protein, namely, WSSV164, which was initially identified from the forward suppression subtractive hybridization (SSH) cDNA library of the PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon that was challenged with white spot syndrome virus (WSSV). Using the yeast two-hybrid system, WSSV164 was found to interact with the PmproPO2 protein. The subsequent validation assay by co-immunoprecipitation revealed that WSSV164 directly bound to both PmproPO1 and PmproPO2. The gene silencing experiment was carried out to explore the role of WSSV164 in the control of the proPO pathway in shrimp, and the results showed that suppression of WSSV164 can restore PO activity in WSSV-infected shrimp hemolymph. The recombinant proteins of PmproPO1 and PmproPO2 were produced in Sf-9 cells and were shown to be successfully activated by exogenous trypsin and endogenous serine proteinases from shrimp hemocyte lysate supernatant (HLS), yielding PO activity in vitro. Moreover, the activated PO activity in shrimp HLS was dose-dependently reduced by the recombinant WSSV164 protein, suggesting that WSSV164 may interfere with the activation of the proPO system in shrimp. Taken together, these results suggest an alternative infection route of WSSV through the encoded viral protein WSSV164 that binds to the PmproPO1 and PmproPO2 proteins, interfering with the activation of the melanization cascade in shrimp.

Two novel totiviruses in the white-backed planthopper, Sogatella furcifera.

There is little information about commensal viruses in the white-backed planthopper, Sogatella furcifera, although it is an important agricultural insect. Here, two novel double-stranded RNA viruses related to the viruses in the family Totiviridae were identified using next-generation sequencing and tentatively named Sogatella furcifera totivirus 1 and 2 (SfTV1 and SfTV2). Their complete genomes consist of 6310 and 6303 nt, respectively, showing typical genomic features with viruses in the family Totiviridae. Identity, phylogenetic and conserved sequence analyses showed that SfTV1, SfTV2 and three other insect viruses may form a proposed novel genus of the family Totiviridae. Vertical transmission of the two viruses was highly efficient, and they were detected in all insect tissues and developmental stages, with the highest titres in the adult and in the haemolymph and reproductive organs. To our knowledge, this is the first report of viruses in the family Totiviridae found in a hemipteran insect.

Purification of virus-like particles (VLPs) expressed in the silkworm Bombyx mori.

Virus-like particles (VLPs) are a promising and developing option for vaccination and gene therapy. They are also interesting as shuttles for drug targeting. Currently, several different gene expression systems are available, among which the silkworm expression system is known for its mass production capacity. However, cost-effective purification with high purity of the target protein is a particular bottleneck for this system. The present review evaluates the advances in the purification of VLPs, especially from silkworm larval hemolymph. Beginning with applicable pre-treatments for VLPs over to chromatography methods and quality control of the purified VLPs. Whereupon the main focus is on the different chromatography approaches for the purification, but the structure of the VLPs and their intended use for humans make also the quality control important. Within this, the stability of the VLPs which has to be considered for the purification is as well discussed.

The Incredible Journey of Begomoviruses in Their Whitefly Vector.

Begomoviruses are vectored in a circulative persistent manner by the whitefly Bemisia tabaci. The insect ingests viral particles with its stylets. Virions pass along the food canal and reach the esophagus and the midgut. They cross the filter chamber and the midgut into the haemolymph, translocate into the primary salivary glands and are egested with the saliva into the plant phloem. Begomoviruses have to cross several barriers and checkpoints successfully, while interacting with would-be receptors and other whitefly proteins. The bulk of the virus remains associated with the midgut and the filter chamber. In these tissues, viral genomes, mainly from the tomato yellow leaf curl virus (TYLCV) family, may be transcribed and may replicate. However, at the same time, virus amounts peak, and the insect autophagic response is activated, which in turn inhibits replication and induces the destruction of the virus. Some begomoviruses invade tissues outside the circulative pathway, such as ovaries and fat cells. Autophagy limits the amounts of virus associated with these organs. In this review, we discuss the different sites begomoviruses need to cross to complete a successful circular infection, the role of the coat protein in this process and the sites that balance between virus accumulation and virus destruction.