Immunofluorescence Assay to Study Early Events of Vector Salivary Gland Colonization by Phytoplasmas.

To visualize phytoplasmas at early stages of vector infection, an immunofluorescence assay was developed. The chapter provides experimental details on dissection of salivary glands, incubation of the dissected organs with phytoplasma suspension, fixation, embedding, sectioning, labeling, and final visualization with confocal microscopy. All the procedure will be described for the leafhopper Euscelidius variegatus, natural vector of "Candidatus phytoplasma asteris" and laboratory vector of Flavescence dorée phytoplasma.

Development of Model Systems for Plant Rhabdovirus Research.

This chapter reviews the discoveries and initial characterizations (1930-1990) of three plant rhabdoviruses, sonchus yellow net virus, potato yellow dwarf virus, and lettuce necrotic yellows virus, that have become model systems for research on this group of enveloped negative-strand RNA plant viruses. We have used our personal perspectives to review the early historical studies of these viruses, the important technologies and tools, such as density gradient centrifugation, that were developed during the research, and to highlight the eminent scientists involved in these discoveries. Early studies on sites of virus replication, virion structure, physicochemical composition, and the use of protoplasts and vector insect cell culture for virus research are discussed, and differences between the nuclear and cytoplasmic lifestyles of plant rhabdoviruses are contrasted. Finally, we briefly summarize the genome organization and more recent developments culminating in the development of a reverse genetics system for plant negative-strand RNA viruses.

Regulation of midgut cell proliferation impacts Aedes aegypti susceptibility to dengue virus.

Aedes aegypti is the vector of some of the most important vector-borne diseases like dengue, chikungunya, zika and yellow fever, affecting millions of people worldwide. The cellular processes that follow a blood meal in the mosquito midgut are directly associated with pathogen transmission. We studied the homeostatic response of the midgut against oxidative stress, as well as bacterial and dengue virus (DENV) infections, focusing on the proliferative ability of the intestinal stem cells (ISC). Inhibition of the peritrophic matrix (PM) formation led to an increase in reactive oxygen species (ROS) production by the epithelial cells in response to contact with the resident microbiota, suggesting that maintenance of low levels of ROS in the intestinal lumen is key to keep ISCs division in balance. We show that dengue virus infection induces midgut cell division in both DENV susceptible (Rockefeller) and refractory (Orlando) mosquito strains. However, the susceptible strain delays the activation of the regeneration process compared with the refractory strain. Impairment of the Delta/Notch signaling, by silencing the Notch ligand Delta using RNAi, significantly increased the susceptibility of the refractory strains to DENV infection of the midgut. We propose that this cell replenishment is essential to control viral infection in the mosquito. Our study demonstrates that the intestinal epithelium of the blood fed mosquito is able to respond and defend against different challenges, including virus infection. In addition, we provide unprecedented evidence that the activation of a cellular regenerative program in the midgut is important for the determination of the mosquito vectorial competence.

Development of leafhopper cell culture to trace the early infection process of a nucleorhabdovirus, rice yellow stunt virus, in insect vector cells.

BACKGROUND: In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear. METHODS: In present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm. RESULTS: Here we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment. CONCLUSIONS: In summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.

In vitro and in vivo host range of Anopheles gambiae densovirus (AgDNV).

AgDNV is a powerful gene transduction tool and potential biological control agent for Anopheles mosquitoes. Using a GFP reporter virus system, we investigated AgDNV host range specificity in four arthropod cell lines (derived from An. gambiae, Aedes albopictus and Drosophila melanogaster) and six mosquito species from 3 genera (An. gambiae, An. arabiensis, An. stephensi, Ae. albopictus, Ae. aegypti and Culex tarsalis). In vitro, efficient viral invasion, replication and GFP expression was only observed in MOS55 An. gambiae cells. In vivo, high levels of GFP were observed in An. gambiae mosquitoes. Intermediate levels of GFP were observed in the closely related species An. arabiensis. Low levels of GFP were observed in An. stephensi, Ae. albopictus, Ae. aegypti and Cx. tarsalis. These results suggest that AgDNV is a specific gene transduction tool for members of the An. gambiae species complex, and could be potentially developed into a biocontrol agent with minimal off-target effects.

Direct Evidence of Egestion and Salivation of Xylella fastidiosa Suggests Sharpshooters Can Be "Flying Syringes".

Xylella fastidiosa is unique among insect-transmitted plant pathogens because it is propagative but noncirculative, adhering to and multiplying on the cuticular lining of the anterior foregut. Any inoculation mechanism for X. fastidiosa must explain how bacterial cells exit the vector's stylets via the food canal and directly enter the plant. A combined egestion-salivation mechanism has been proposed to explain these unique features. Egestion is the putative outward flow of fluid from the foregut via hypothesized bidirectional pumping of the cibarium. The present study traced green fluorescent protein-expressing X. fastidiosa or fluorescent nanoparticles acquired from artificial diets by glassy-winged sharpshooters, Homalodisca vitripennis, as they were egested into simultaneously secreted saliva. X. fastidiosa or nanoparticles were shown to mix with gelling saliva to form fluorescent deposits and salivary sheaths on artificial diets, providing the first direct, conclusive evidence of egestion by any hemipteran insect. Therefore, the present results strongly support an egestion-salivation mechanism of X. fastidiosa inoculation. Results also support that a column of fluid is transiently held in the foregut without being swallowed. Evidence also supports (but does not definitively prove) that bacteria were suspended in the column of fluid during the vector's transit from diet to diet, and were egested with the held fluid. Thus, we hypothesize that sharpshooters could be true "flying syringes," especially when inoculation occurs very soon after uptake of bacteria, suggesting the new paradigm of a nonpersistent X. fastidiosa transmission mechanism.

Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference.

The protein synthesis machineries of two distinct phyla of the Animal kingdom, insects of Arthropoda and mammals of Chordata, have different preferences for how to best encode proteins. Nevertheless, arboviruses (arthropod-borne viruses) are capable of infecting both mammals and insects just like arboviruses that use insect vectors to infect plants. These organisms have evolved carefully balanced genomes that can efficiently use the translational machineries of different phyla, even if the phyla belong to different kingdoms. Using dengue virus as an example, we have undone the genome encoding balance and specifically shifted the encoding preference away from mammals. These mammalian-attenuated viruses grow to high titers in insect cells but low titers in mammalian cells, have dramatically increased LD50s in newborn mice, and induce high levels of protective antibodies. Recoded arboviruses with a bias toward phylum-specific expression could form the basis of a new generation of live attenuated vaccine candidates.

Localization and distribution of wheat dwarf virus in its vector leafhopper, Psammotettix alienus.

Numerous virus pathogens are transmitted by specific arthropod vectors. Understanding the mechanism of transmission is a critical step in the epidemiology of plant viruses and is crucial for the development of effective disease control strategies. In this study, we describe the localization and distribution of Wheat dwarf virus (WDV), an economically important and widespread single-stranded DNA virus, in its leafhopper vector, Psammotettix alienus. The results suggest that WDV not only can move to the salivary glands from the anterior and middle midgut via the hemocoel but also can pass directly through the sheath of the filter chamber and be readily transmitted to healthy wheat plants within 5 min of an acquisition access period on infected plants. When a bacterial-expressed recombinant capsid protein (CP) was incubated with the internal organs of leafhoppers, CP-immunoreactive antigens were found at the anterior and middle midgut. Furthermore, when leafhoppers were fed with an antiserum raised against the CP, the accumulation of WDV in the gut cells, hemocoel, and salivary glands was significantly reduced. These data provide evidence that transmission of WDV is determined by a CP-mediated virion-vector retention mechanism.

Transmission of Pantoea ananatis and P. agglomerans, causal agents of center rot of onion (Allium cepa), by onion thrips (Thrips tabaci) through feces.

Frankliniella fusca, the tobacco thrips, has been shown to acquire and transmit Pantoea ananatis, one of the causal agents of the center rot of onion. Although Thrips tabaci, the onion thrips, is a common pest of onions, its role as a vector of P. ananatis has been unknown. The bacterium, P. agglomerans, is also associated with the center rot of onion, but its transmission by thrips has not been previously investigated. In this study, we investigated the relationship of T. tabaci with P. ananatis and P. agglomerans. Surface-sterilized T. tabaci were provided with various acquisition access periods (AAP) on onion leaves inoculated with either P. ananatis or P. agglomerans. A positive exponential relationship was observed between thrips AAP duration and P. ananatis (R² = 0.967; P = 0.023) or P. agglomerans acquisition (R² = 0.958; P = 0.017). Transmission experiments conducted with T. tabaci adults indicated that 70% of the seedlings developed center rot symptoms 15 days after inoculation. Immunofluorescence microscopy with antibodies specific to P. ananatis revealed that the bacterium was localized only in the gut of T. tabaci adults. Mechanical inoculation of onion seedlings with fecal rinsates alone produced center rot but not with salivary secretions. Together these results suggested that T. tabaci could efficiently transmit P. ananatis and P. agglomerans.

Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate-type interactions.

Spiroplamas are helical, cell wall-less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram-positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin-less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface-exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild-type but not of the spiralin-less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin.

Chagas disease: control, elimination and eradication. Is it possible?

From an epidemiological point of view, Chagas disease and its reservoirs and vectors can present the following characteristics: (i) enzooty, maintained by wild animals and vectors, with broad occurrence from southern United States of America (USA) to southern Argentina and Chile (42ºN 49ºS), (ii) anthropozoonosis, when man invades the wild ecotope and becomes infected with Trypanosoma cruzi from wild animals or vectors or when the vectors and wild animals, especially marsupials, invade the human domicile and infect man, (iii) zoonosis-amphixenosis and exchanged infection between animals and humans by domestic vectors in endemic areas and (iv) zooanthroponosis, infection that is transmitted from man to animals, by means of domestic vectors, which is the rarest situation in areas endemic for Chagas disease. The characteristics of Chagas disease as an enzooty of wild animals and as an anthropozoonosis are seen most frequently in the Brazilian Amazon and in the Pan-Amazon region as a whole, where there are 33 species of six genera of wild animals: Marsupialia, Chiroptera, Rodentia, Edentata (Xenarthra), Carnivora and Primata and 27 species of triatomines, most of which infected with T. cruzi . These conditions place the resident populations of this area or its visitors - tourists, hunters, fishermen and especially the people whose livelihood involves plant extraction - at risk of being affected by Chagas disease. On the other hand, there has been an exponential increase in the acute cases of Chagas disease in that region through oral transmission of T. cruzi , causing outbreaks of the disease. In four seroepidemiological surveys that were carried out in areas of the microregion of the Negro River, state of Amazonas, in 1991, 1993, 1997 and 2010, we found large numbers of people who were serologically positive for T. cruzi infection. The majority of them and/or their relatives worked in piassava extraction and had come into contact with and were stung by wild triatomines in that area. Finally, a characteristic that is greatly in evidence currently is the migration of people with Chagas disease from endemic areas of Latin America to non-endemic countries. This has created a new dilemma for these countries: the risk of transmission through blood transfusion and the onus of controlling donors and treating migrants with the disease. As an enzooty of wild animals and vectors, and as an anthropozoonosis, Chagas disease cannot be eradicated, but it must be controlled by transmission elimination to man.

Artificial activation of mature unfertilized eggs in the malaria vector mosquito, Anopheles stephensi (Diptera, Culicidae).

In the past decade, many transgenic lines of mosquitoes have been generated and analyzed, whereas the maintenance of a large number of transgenic lines requires a great deal of effort and cost. In vitro fertilization by an injection of cryopreserved sperm into eggs has been proven to be effective for the maintenance of strains in mammals. The technique of artificial egg activation is a prerequisite for the establishment of in vitro fertilization by sperm injection. We demonstrated that artificial egg activation is feasible in the malaria vector mosquito, Anopheles stephensi (Diptera, Culicidae). Nearly 100% of eggs dissected from virgin females immersed in distilled water darkened, similar to normally oviposited fertilized eggs. It was revealed by the cytological examination of chromosomes that meiotic arrest was relieved in these eggs approximately 20 min after incubation in water. Biochemical examinations revealed that MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated protein kinase) and MEK (MAPK/ERK kinase) were dephosphorylated similar to that in fertilized eggs. These results indicate that dissected unfertilized eggs were activated in distilled water and started development. Injection of distilled water into body cavity of the virgin blood-fed females also induced activation of a portion of eggs in the ovaries. The technique of artificial egg activation is expected to contribute to the success of in vitro fertilization in A. stephensi.

wFlu: characterization and evaluation of a native Wolbachia from the mosquito Aedes fluviatilis as a potential vector control agent.

There is currently considerable interest and practical progress in using the endosymbiotic bacteria Wolbachia as a vector control agent for human vector-borne diseases. Such vector control strategies may require the introduction of multiple, different Wolbachia strains into target vector populations, necessitating the identification and characterization of appropriate endosymbiont variants. Here, we report preliminary characterization of wFlu, a native Wolbachia from the neotropical mosquito Aedes fluviatilis, and evaluate its potential as a vector control agent by confirming its ability to cause cytoplasmic incompatibility, and measuring its effect on three parameters determining host fitness (survival, fecundity and fertility), as well as vector competence (susceptibility) for pathogen infection. Using an aposymbiotic strain of Ae. fluviatilis cured of its native Wolbachia by antibiotic treatment, we show that in its natural host wFlu causes incomplete, but high levels of, unidirectional cytoplasmic incompatibility, has high rates of maternal transmission, and no detectable fitness costs, indicating a high capacity to rapidly spread through host populations. However, wFlu does not inhibit, and even enhances, oocyst infection with the avian malaria parasite Plasmodium gallinaceum. The stage- and sex-specific density of wFlu was relatively low, and with limited tissue distribution, consistent with the lack of virulence and pathogen interference/symbiont-mediated protection observed. Unexpectedly, the density of wFlu was also shown to be specifically-reduced in the ovaries after bloodfeeding Ae. fluviatilis. Overall, our observations indicate that the Wolbachia strain wFlu has the potential to be used as a vector control agent, and suggests that appreciable mutualistic coevolution has occurred between this endosymbiont and its natural host. Future work will be needed to determine whether wFlu has virulent host effects and/or exhibits pathogen interference when artificially-transfected to the novel mosquito hosts that are the vectors of human pathogens.

Transfer of Escherichia coli O157:H7 to spinach by house flies, Musca domestica (Diptera: Muscidae).

Filth flies are known mechanical vectors of pathogenic bacteria in hospital and restaurant settings, but their role as vectors for disseminating microbes to plants has not been demonstrated. Escherichia coli O157:H7 deposition by flies onto spinach was studied using molecular, microbiological, and microscopy techniques. Relative quantitative polymerase chain reaction studies showed that bacteria acquired by flies from contaminated cattle manure and deposited in regurgitation spots on leaves survived and multiplied. Scanning electron microscopy of the regurgitation spots of flies exposed to manure inoculated with E. coli suggested the multiplication of bacteria-like organisms within the spots. This finding implies that the bacteria were active and is consistent with a hypothesis that regurgitation spots serve as a nutrition source allowing E. coli O157:H7 to survive on the spinach phylloplane. E. coli O157:H7 persisted on fly body surfaces up to 13 days after exposure to acquisition sources, suggesting that fly cuticular surfaces are conducive to the growth of this pathogen. These results are consistent with the hypothesis of bioenhanced transmission of human pathogens by house flies and suggest that filth flies may affect the microbial safety of fresh produce.

Chagas disease: control, elimination and eradication. Is it possible?

From an epidemiological point of view, Chagas disease and its reservoirs and vectors can present the following characteristics: (i) enzooty, maintained by wild animals and vectors, with broad occurrence from southern United States of America (USA) to southern Argentina and Chile (42ºN 49ºS), (ii) anthropozoonosis, when man invades the wild ecotope and becomes infected with Trypanosoma cruzi from wild animals or vectors or when the vectors and wild animals, especially marsupials, invade the human domicile and infect man, (iii) zoonosis-amphixenosis and exchanged infection between animals and humans by domestic vectors in endemic areas and (iv) zooanthroponosis, infection that is transmitted from man to animals, by means of domestic vectors, which is the rarest situation in areas endemic for Chagas disease. The characteristics of Chagas disease as an enzooty of wild animals and as an anthropozoonosis are seen most frequently in the Brazilian Amazon and in the Pan-Amazon region as a whole, where there are 33 species of six genera of wild animals: Marsupialia, Chiroptera, Rodentia, Edentata (Xenarthra), Carnivora and Primata and 27 species of triatomines, most of which infected with T. cruzi . These conditions place the resident populations of this area or its visitors - tourists, hunters, fishermen and especially the people whose livelihood involves plant extraction - at risk of being affected by Chagas disease. On the other hand, there has been an exponential increase in the acute cases of Chagas disease in that region through oral transmission of T. cruzi , causing outbreaks of the disease. In four seroepidemiological surveys that were carried out in areas of the microregion of the Negro River, state of Amazonas, in 1991, 1993, 1997 and 2010, we found large numbers of people who were serologically positive for T. cruzi infection. The majority of them and/or their relatives worked in piassava extraction and had come into contact with and were stung by wild triatomines in that area. Finally, a characteristic that is greatly in evidence currently is the migration of people with Chagas disease from endemic areas of Latin America to non-endemic countries. This has created a new dilemma for these countries: the risk of transmission through blood transfusion and the onus of controlling donors and treating migrants with the disease. As an enzooty of wild animals and vectors, and as an anthropozoonosis, Chagas disease cannot be eradicated, but it must be controlled by transmission elimination to man.

Transcriptome and comparative gene expression analysis of Sogatella furcifera (Horváth) in response to southern rice black-streaked dwarf virus.

BACKGROUND: The white backed planthopper (WBPH), Sogatella furcifera (Horváth), causes great damage to many crops by direct feeding or transmitting plant viruses. Southern rice black-streaked dwarf virus (SRBSDV), transmitted by WBPH, has become a great threat to rice production in East Asia. METHODOLOGY/PRINCIPAL FINDINGS: By de novo transcriptome assembling and massive parallel pyrosequencing, we constructed two transcriptomes of WBPH and profiled the alternation of gene expression in response to SRBSDV infection in transcriptional level. Over 25 million reads of high-quality DNA sequences and 81388 different unigenes were generated using Illumina technology from both viruliferous and non-viruliferous WBPH. WBPH has a very similar gene ontological distribution to other two closely related rice planthoppers, Nilaparvata lugens and Laodelphax striatellus. 7291 microsatellite loci were also predicted which could be useful for further evolutionary analysis. Furthermore, comparative analysis of the two transcriptomes generated from viruliferous and non-viruliferous WBPH provided a list of candidate transcripts that potentially were elicited as a response to viral infection. Pathway analyses of a subset of these transcripts indicated that SRBSDV infection may perturb primary metabolism and the ubiquitin-proteasome pathways. In addition, 5.5% (181 out of 3315) of the genes in cell cytoskeleton organization pathway showed obvious changes. Our data also demonstrated that SRBSDV infection activated the immunity regulatory systems of WBPH, such as RNA interference, autophagy and antimicrobial peptide production. CONCLUSIONS/SIGNIFICANCE: We employed massively parallel pyrosequencing to collect ESTs from viruliferous and non-viruliferous samples of WBPH. 81388 different unigenes have been obtained. We for the first time described the direct effects of a Reoviridae family plant virus on global gene expression profiles of its insect vector using high-throughput sequencing. Our study will provide a road map for future investigations of the fascinating interactions between Reoviridae viruses and their insect vectors, and provide new strategies for crop protection.

Wolbachia infection and mitochondrial DNA comparisons among Culex mosquitoes in South West Iran.

The control of mosquito borne diseases needs new methods given widespread insecticide resistance in many mosquito species. The inherited endosymbiont Wolbachia, found in many arthropods, provides a biological system to reduce the transmission of these diseases and replace the population of vectors with non-vectors using cytoplasmic incompatibility. The aim of this study was to understand the rate of Wolbachia infection among Culex species in the region and to see the effect of Wolbachia infection on mitochondrial genome. In this study three species of Culex mosquitoes were collected from Shoushtar in south west of Iran and examined for Wolbachia infection by Polymerase Chain Reaction (PCR). All of the C. quinquefasciatus specimens were infected with Wolbachia, while C. tritaeniorynchus and C. theileri showed no infection with Wolbachia. The 340 bp of AT rich of mtDNA was sequenced from 30 individuals, 10 individuals of each species. Three sequence haplotypes were found in C. tritaeniorynchus and C. theileri while there was only one haplotype in C. quinquefasciatus. The reduction of haplotypes diversity may be result of a sweep of Wolbachia in this species.

Effects of manipulating apoptosis on Sindbis virus infection of Aedes aegypti mosquitoes.

Improved control of vector-borne diseases requires an understanding of the molecular factors that determine vector competence. Apoptosis has been shown to play a role in defense against viruses in insects and mammals. Although some observations suggest a correlation between apoptosis and resistance to arboviruses in mosquitoes, there is no direct evidence tying apoptosis to arbovirus vector competence. To determine whether apoptosis can influence arbovirus replication in mosquitoes, we manipulated apoptosis in Aedes aegypti mosquitoes by silencing the expression of genes that either positively or negatively regulate apoptosis. Silencing of the A. aegypti anti-apoptotic gene iap1 (Aeiap1) caused apoptosis in midgut epithelium, alterations in midgut morphology, and 60 to 70% mosquito mortality. Mortality induced by Aeiap1 silencing was rescued by cosilencing the initiator caspase gene Aedronc, indicating that the mortality was due to apoptosis. When mosquitoes which had been injected with Aeiap1 double-stranded RNA (dsRNA) were orally infected with Sindbis virus (SINV), increased midgut infection and virus dissemination to other organs were observed. This increase in virus infection may have been due to the effects of widespread apoptosis on infection barriers or innate immunity. In contrast, silencing the expression of Aedronc, which would be expected to inhibit apoptosis, reduced SINV midgut infection and virus dissemination. Thus, our data suggest that some level of caspase activity and/or apoptosis may be necessary for efficient virus replication and dissemination in mosquitoes. This is the first study to directly test the roles of apoptosis and caspases in determining mosquito vector competence for arboviruses.

Chikungunya virus infection of cell lines: analysis of the East, Central and South African lineage.

Chikungunya virus (CHIKV) is a re-emerging mosquito borne alphavirus that has caused large scale epidemics in the countries around the Indian Ocean, as well as leading to autochthonous transmission in some European countries. The transmission of the disease has been driven by the emergence of an African lineage of CHIKV with enhanced transmission and dissemination in Aedes mosquito hosts. Two main genotypes of this lineage have been circulating, characterized by the presence of a substitution of a valine for an alanine at position 226 of the E1 protein. The outbreak, numbering in millions of cases in the infected areas, has been associated with increasing numbers of cases with non-classical presentation including encephalitis and meningitis. This study sought to compare the original Ross strain with two isolates from the recent outbreak of chikungunya fever in respect of infectivity and the induction of apoptosis in eight mammalian cell lines and two insect cell lines, in addition to generating a comprehensive virus production profile for one of the newer isolates. Results showed that in mammalian cells there were few differences in either tropism or pathogenicity as assessed by induction of apoptosis with the exception of Hela cells were the recent valine isolate showed less infectivity. The Aedes albopictus C6/36 cell line was however significantly more permissive for both of the more recent isolates than the Ross strain. The results suggest that the increased infectivity seen in insect cells derives from an evolution of the CHIKV genome not solely associated with the E1:226 substitution.

Involvement of sulfated glycosaminoglycans on the development and attachment of Trypanosoma cruzi to the luminal midgut surface in the vector, Rhodnius prolixus.

In the present study, we investigated the involvement of sulfated glycosaminoglycans in both the in vivo development and adhesion of T. cruzi epimastigotes to the luminal surface of the digestive tract of the insect vector, Rhodnius prolixus. Pre-incubation of T. cruzi, Dm 28c epimastigotes with heparin, chondroitin 4-sulfate, chondroitin 6-sulfate or protamine chloridrate inhibited in vitro attachment of parasites to the insect midgut. Enzymatic removal of heparan sulfate moieties by heparinase I or of chondroitin sulfate moieties by chondroitinase AC from the insect posterior midgut abolished epimastigote attachment in vitro. These treatments also reduced the labelling of anionic sites exposed at the luminal surface of the perimicrovillar membranes in the triatomine midgut epithelial cells. Inclusion of chondroitin 4-sulfate or chondroitin 6-sulfate and to a lesser extent, heparin, in the T. cruzi-infected bloodmeal inhibited the establishment of parasites in R. prolixus. These observations indicate that sulfated glycosaminoglycans are one of the determinants for both adhesion of the T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine and the parasite infection in the insect vector, R. prolixus.