Multifaceted contributions of Dicer2 to arbovirus transmission by Aedes aegypti.

Arthropod-borne viruses (arboviruses) transmitted by Aedes aegypti mosquitoes are an increasing threat to global health. The small interfering RNA (siRNA) pathway is considered the main antiviral immune pathway of insects, but its effective impact on arbovirus transmission is surprisingly poorly understood. Here, we use CRISPR-Cas9-mediated gene editing in vivo to mutate Dicer2, a gene encoding the RNA sensor and key component of the siRNA pathway. The loss of Dicer2 enhances early viral replication and systemic viral dissemination of four medically significant arboviruses (chikungunya, Mayaro, dengue, and Zika viruses) representing two viral families. However, Dicer2 mutants and wild-type mosquitoes display overall similar levels of vector competence. In addition, Dicer2 mutants undergo significant virus-induced mortality during infection with chikungunya virus. Together, our results define a multifaceted role for Dicer2 in the transmission of arboviruses by Ae. aegypti mosquitoes and pave the way for further mechanistic investigations.

Recent advances in the development of methyltransferase (MTase) inhibitors against (re)emerging arboviruses diseases dengue and Zika.

Emerging and/or re-emerging viral diseases such as dengue and Zika are a worldwide concern. Therefore, new antiviral therapeutics are necessary. In this sense, a non-structural protein with methyltransferase (MTase) activity is an attractive drug target because it plays a crucial role in dengue and Zika virus replication. Different drug strategies such as virtual screening, molecular docking, and molecular dynamics have identified new inhibitors that bind on the MTase active site. Therefore, in this review, we analyze MTase inhibitors, including S-adenosyl-L-methionine (SAM), S-adenosyl-l-homocysteine (SAH) and guanosine-5'-triphosphate (GTP) analogs, nitrogen-containing heterocycles (pyrimidine, adenosine, and pyridine), urea derivatives, and natural products. Advances in the design of MTase inhibitors could lead to the optimization of a possible single or broad-spectrum antiviral drug against dengue and Zika virus.

Mosquito saliva enhances virus infection through sialokinin-dependent vascular leakage.

Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.

GrpE is involved in mitochondrial function and is an effective target for RNAi-mediated pest and arbovirus control.

Laodelphax striatellus is a sap-feeding pest and the main insect vector of rice stripe virus (RSV). There is an urgent need to identify molecular targets to control this insect pest and plant arboviruses. In this study, we identified a L. striatellus gene (named LsGrpE) encoding a GroP-E-like protein. We found that the LsGrpE protein localized to mitochondria. Using gene-specific dsRNA to interfere with the expression of LsGrpE led to a significant increase in insect mortality, and most of the surviving insects could not develop into adults. Further analyses revealed that LsGrpE deficiency caused mitochondrial dysfunction and inhibited the insulin pathway, resulting in diabetes-like symptoms such as elevated blood sugar, inactive behaviour, developmental delay, and death. In addition, LsGrpE deficiency significantly reduced the RSV titre in surviving L. striatellus, and indirectly prevented viral vertical transmission by reducing the number of adults. We generated transgenic rice plants expressing LsGrpE-specific dsRNA, and the dsRNA was acquired by L. striatellus during feeding, resulting in increased insect mortality and the prevention of arboviral transmission. This study clarifies the function of LsGrpE and demonstrates that LsGrpE can be used as a molecular target of plant-generated dsRNA to resist this sap-feeding pest, a17nd therefore, its transmitted arboviruses.

Microclimate and the vertical stratification of potential bridge vectors of mosquito­borne viruses captured by nets and ovitraps in a central Amazonian forest bordering Manaus, Brazil.

In the Americas, some mosquito-borne viruses such as Zika, chikungunya, and dengue circulate among humans in urban transmission cycles, while others, including yellow fever and Mayaro, circulate among monkeys in sylvatic cycles. The intersection of humans and wildlife at forest edges creates risk for zoonotic virus exchange. We built a scaffold tower at the edge of a treefall gap in rainforest bordering Manaus, Brazil, to identify vectors that may bridge transmission between humans and monkeys. We vertically sampled diurnally active, anthropophilic mosquitoes using handheld nets at 0, 5, and 9 m and container-breeding mosquitoes in ovitraps at 0, 5, 10, and 15 m. Haemagogus janthinomys and Psorophora amazonica were present in high relative abundance in nets at each height sampled, while anthropophilic species were uncommon in ovitraps. Hg. janthinomys was more abundant at elevated heights than at ground level, while Ps. amazonica abundance was not significantly stratified across heights. The presence of each species increased with increasing 7-day rainfall lagged at 1 week, and at 1 and 4 weeks prior to collection, respectively. In addition, Hg. janthinomys was most frequently collected at 29.9 °C, irrespective of height. These data provide insight into the potential role of each species as bridge vectors.

Novel Symbiotic Genome-Scale Model Reveals Wolbachia's Arboviral Pathogen Blocking Mechanism in Aedes aegypti.

Wolbachia are endosymbiont bacteria known to infect arthropods causing different effects, such as cytoplasmic incompatibility and pathogen blocking in Aedes aegypti. Although several Wolbachia strains have been studied, there is little knowledge regarding the relationship between this bacterium and their hosts, particularly on their obligate endosymbiont nature and its pathogen blocking ability. Motivated by the potential applications on disease control, we developed a genome-scale model of two Wolbachia strains: wMel and the strongest Dengue blocking strain known to date: wMelPop. The obtained metabolic reconstructions exhibit an energy metabolism relying mainly on amino acids and lipid transport to support cell growth that is consistent with altered lipid and cholesterol metabolism in Wolbachia-infected mosquitoes. The obtained metabolic reconstruction was then coupled with a reconstructed mosquito model to retrieve a symbiotic genome-scale model accounting for 1,636 genes and 6,408 reactions of the Aedes aegypti-Wolbachia interaction system. Simulation of an arboviral infection in the obtained novel symbiotic model represents a metabolic scenario characterized by pathogen blocking in higher titer Wolbachia strains, showing that pathogen blocking by Wolbachia infection is consistent with competition for lipid and amino acid resources between arbovirus and this endosymbiotic bacteria. IMPORTANCE Arboviral diseases such as Zika and Dengue have been on the rise mainly due to climate change, and the development of new treatments and strategies to limit their spreading is needed. The use of Wolbachia as an approach for disease control has motivated new research related to the characterization of the mechanisms that underlie its pathogen-blocking properties. In this work, we propose a new approach for studying the metabolic interactions between Aedes aegypti and Wolbachia using genome-scale models, finding that pathogen blocking is mainly influenced by competition for the resources required for Wolbachia and viral replication.

aBravo Is a Novel Aedes aegypti Antiviral Protein that Interacts with, but Acts Independently of, the Exogenous siRNA Pathway Effector Dicer 2.

Mosquitoes, such as Aedes aegypti, can transmit arboviruses to humans. The exogenous short interfering RNA (exo-siRNA) pathway plays a major antiviral role in controlling virus infection in mosquito cells. The Dicer 2 (Dcr2) nuclease is a key effector protein in this pathway, which cleaves viral double-stranded RNA into virus-derived siRNAs that are further loaded onto an effector called Argonaute 2 (Ago2), which as part of the multiprotein RNA-induced silencing complex (RISC) targets and cleaves viral RNA. In order to better understand the effector protein Dcr2, proteomics experiments were conducted to identify interacting cellular partners. We identified several known interacting partners including Ago2, as well as two novel and previously uncharacterized Ae. aegypti proteins. The role of these two proteins was further investigated, and their interactions with Dcr2 verified by co-immunoprecipitation. Interestingly, despite their ability to interact with Ago2 and Piwi4, neither of these proteins was found to affect exo-siRNA silencing in a reporter assay. However, one of these proteins, Q0IFK9, subsequently called aBravo (aedine broadly active antiviral protein), was found to mediate antiviral activity against positive strand RNA arboviruses. Intriguingly the presence of Dcr2 was not necessary for this effect, suggesting that this interacting antiviral effector may act as part of protein complexes with potentially separate antiviral activities.

Nanostructured impedimetric lectin-based biosensor for arboviruses detection.

Arboviruses have been emerging as a significant global health problem due to the recurrent epidemics. Arboviruses require the development of new diagnostic devices due to the nonspecific clinical manifestations. Herein, we report a biosensor based on cysteine (Cys), zinc oxide nanoparticles (ZnONp), and Concanavalin A (ConA) lectin to differentiate between arboviruses infections. ConA is capable of interacting with the saccharide components of the viral capsid. In this study, we evaluated the reproducibility, sensitivity, and specificity of the sensor for the virus of Dengue type 2 (DENV2), Zika (ZIKV), Chikungunya (CHIKV), and Yellow fever (YFV). Atomic force microscopy measurements confirmed the electrode surface modification and revealed a heterogeneous topography during the biorecognition process. Cyclic voltammetry (CV) and impedance spectroscopy (EIS) were used to characterize the biosensor. The blockage of the oxidation-reduction process is related to the formation of Cys-ZnONp-ConA system on the electroactive area and its subsequent interaction with viral glycoproteins. The sensor exhibited a linear response to different concentrations of the studied arboviruses. Our study demonstrates that ConA lectin recognizes the structural glycoproteins of the DENV2, ZIKV, CHIKV, and YFV. DENV2 is the most structurally similar to ZIKV. Our results have shown that the impedimetric response correlates with the structural glycoproteins, as follow: DENV2 (18.6 kΩ) > ZIKV (14.6 kΩ) > CHIKV (6.86 kΩ) > YFV (5.98 kΩ). The homologous structural regions contribute to ConA-arboviruses recognition. Our results demonstrate the use of the proposed system for the development of biosensors for arboviruses infections.

2019 meeting of the global virus network.

The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 52 GVN Centers of Excellence and 9 Affiliate laboratories in 32 countries. The 11th International GVN meeting was held from June 9-11, 2019 in Barcelona, Spain and was jointly organized with the Spanish Society of Virology. A common theme throughout the meeting was globalization and climate change. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including severe virus epidemics, anticipation and preparedness for changing disease dynamics, host-pathogen interactions, zoonotic virus infections, ethical preparedness for epidemics and pandemics, one health and antivirals.

Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo.

Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses.IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.

The roles of apoptosis, autophagy and unfolded protein response in arbovirus, influenza virus, and HIV infections.

Virus infection induces different cellular responses in infected cells. These include cellular stress responses like autophagy and unfolded protein response (UPR). Both autophagy and UPR are connected to programed cell death I (apoptosis) in chronic stress conditions to regulate cellular homeostasis via Bcl2 family proteins, CHOP and Beclin-1. In this review article we first briefly discuss arboviruses, influenza virus, and HIV and then describe the concepts of apoptosis, autophagy, and UPR. Finally, we focus upon how apoptosis, autophagy, and UPR are involved in the regulation of cellular responses to arboviruses, influenza virus and HIV infections. Abbreviation: AIDS: Acquired Immunodeficiency Syndrome; ATF6: Activating Transcription Factor 6; ATG6: Autophagy-specific Gene 6; BAG3: BCL Associated Athanogene 3; Bak: BCL-2-Anatagonist/Killer1; Bax; BCL-2: Associated X protein; Bcl-2: B cell Lymphoma 2x; BiP: Chaperon immunoglobulin heavy chain binding Protein; CARD: Caspase Recruitment Domain; cART: combination Antiretroviral Therapy; CCR5: C-C Chemokine Receptor type 5; CD4: Cluster of Differentiation 4; CHOP: C/EBP homologous protein; CXCR4: C-X-C Chemokine Receptor Type 4; Cyto c: Cytochrome C; DCs: Dendritic Cells; EDEM1: ER-degradation enhancing-a-mannosidase-like protein 1; ENV: Envelope; ER: Endoplasmic Reticulum; FasR: Fas Receptor;G2: Gap 2; G2/M: Gap2/Mitosis; GFAP: Glial Fibrillary Acidic Protein; GP120: Glycoprotein120; GP41: Glycoprotein41; HAND: HIV Associated Neurodegenerative Disease; HEK: Human Embryonic Kidney; HeLa: Human Cervical Epithelial Carcinoma; HIV: Human Immunodeficiency Virus; IPS-1: IFN-ß promoter stimulator 1; IRE-1: Inositol Requiring Enzyme 1; IRGM: Immunity Related GTPase Family M protein; LAMP2A: Lysosome Associated Membrane Protein 2A; LC3: Microtubule Associated Light Chain 3; MDA5: Melanoma Differentiation Associated gene 5; MEF: Mouse Embryonic Fibroblast; MMP: Mitochondrial Membrane Permeabilization; Nef: Negative Regulatory Factor; OASIS: Old Astrocyte Specifically Induced Substrate; PAMP: Pathogen-Associated Molecular Pattern; PERK: Pancreatic Endoplasmic Reticulum Kinase; PRR: Pattern Recognition Receptor; Puma: P53 Upregulated Modulator of Apoptosis; RIG-I: Retinoic acid-Inducible Gene-I; Tat: Transactivator Protein of HIV; TLR: Toll-like receptor; ULK1: Unc51 Like Autophagy Activating Kinase 1; UPR: Unfolded Protein Response; Vpr: Viral Protein Regulatory; XBP1: X-Box Binding Protein 1.

Arbovirus lifecycle in mosquito: acquisition, propagation and transmission.

Mosquitoes are haematophagous vectors for hundreds of pathogenic viruses that are aetiological agents of human diseases. In nature, mosquito-borne viruses maintain a lifecycle between mosquitoes and vertebrate animals. Viruses are acquired by a naive mosquito from an infected host by blood meals and then propagate extensively in the mosquito's tissues. This mosquito then becomes a virus reservoir and is competent to transmit the viruses to a naive vertebrate host through the next blood meal. To survive in and efficiently cycle between two distinct host environments, mosquito-borne viruses have evolved delicate and smart strategies to comprehensively exploit host and vector factors. Here, we provide an update on recent studies of the mechanisms of virus survival in, acquisition and transmission by mosquitoes.

Atovaquone Inhibits Arbovirus Replication through the Depletion of Intracellular Nucleotides.

Arthropod-borne viruses represent a significant public health threat worldwide, yet there are few antiviral therapies or prophylaxes targeting these pathogens. In particular, the development of novel antivirals for high-risk populations such as pregnant women is essential to prevent devastating disease such as that which was experienced with the recent outbreak of Zika virus (ZIKV) in the Americas. One potential avenue to identify new and pregnancy-acceptable antiviral compounds is to repurpose well-known and widely used FDA-approved drugs. In this study, we addressed the antiviral role of atovaquone, an FDA Pregnancy Category C drug and pyrimidine biosynthesis inhibitor used for the prevention and treatment of parasitic infections. We found that atovaquone was able to inhibit ZIKV and chikungunya virus virion production in human cells and that this antiviral effect occurred early during infection at the initial steps of viral RNA replication. Moreover, we were able to complement viral replication and virion production with the addition of exogenous pyrimidine nucleosides, indicating that atovaquone functions through the inhibition of the pyrimidine biosynthesis pathway to inhibit viral replication. Finally, using an ex vivo human placental tissue model, we found that atovaquone could limit ZIKV infection in a dose-dependent manner, providing evidence that atovaquone may function as an antiviral in humans. Taken together, these studies suggest that atovaquone could be a broad-spectrum antiviral drug and a potential attractive candidate for the prophylaxis or treatment of arbovirus infection in vulnerable populations, such as pregnant women and children.IMPORTANCE The ability to protect vulnerable populations such as pregnant women and children from Zika virus and other arbovirus infections is essential to preventing the devastating complications induced by these viruses. One class of antiviral therapies may lie in known pregnancy-acceptable drugs that have the potential to mitigate arbovirus infections and disease, yet this has not been explored in detail. In this study, we show that the common antiparasitic drug atovaquone inhibits arbovirus replication through intracellular nucleotide depletion and can impair ZIKV infection in an ex vivo human placental explant model. Our study provides a novel function for atovaquone and highlights that the rediscovery of pregnancy-acceptable drugs with potential antiviral effects can be the key to better addressing the immediate need for treating viral infections and preventing potential birth complications and future disease.

Lack of serological and molecular evidence of arbovirus infections in bats from Brazil.

Viruses are important agents of emerging zoonoses and are a substantial public health issue. Among emerging viruses, an important group are arboviruses, which are characterized by being maintained in nature in cycles involving hematophagous arthropod vectors and a wide range of vertebrate hosts. Recently, bats have received increasing attention as an important source for the emergence of zoonoses and as possible viral reservoirs. Among the arboviruses, there are many representatives of the genera Flavivirus and Alphavirus, which are responsible for important epidemics such as Dengue virus, Zika virus and Chikungunya virus. Due to the importance of analyzing potential viral reservoirs for zoonosis control and expanding our knowledge of bat viruses, this study aimed to investigate the presence of viruses of the Alphavirus and Flavivirus genera in bats. We analyzed serum, liver, lungs and intestine from 103 bats sampled in northeast and southern Brazil via Nested-PCR and the hemagglutination inhibition test. All samples tested in this study were negative for arboviruses, suggesting that no active or past infection was present in the captured bats. These data indicate that the bats examined herein probably do not constitute a reservoir for these viruses in the studied areas. Further studies are needed to clarify the role of bats as reservoirs and sources of infection of these viral zoonoses.

Blood meal acquisition enhances arbovirus replication in mosquitoes through activation of the GABAergic system.

Mosquitoes are hematophagous insects that carry-on and transmit many human viruses. However, little information is available regarding the common mechanisms underlying the infection of mosquitoes by these viruses. In this study, we reveal that the hematophagous nature of mosquitoes contributes to arboviral infection after a blood meal, which suppresses antiviral innate immunity by activating the GABAergic pathway. dsRNA-mediated interruption of the GABA signaling and blockage of the GABAA receptor by the specific inhibitors both significantly impaired arbovirus replication. Consistently, inoculation of GABA enhanced arboviral infection, indicating that GABA signaling facilitates the arboviral infection of mosquitoes. The ingestion of blood by mosquitoes resulted in robust GABA production from glutamic acid derived from blood protein digestion. The oral introduction of glutamic acid increased virus acquisition by mosquitoes via activation of the GABAergic system. Our study reveals that blood meals enhance arbovirus replication in mosquitoes through activation of the GABAergic system.

Comparative genomics shows that viral integrations are abundant and express piRNAs in the arboviral vectors Aedes aegypti and Aedes albopictus.

BACKGROUND: Arthropod-borne viruses (arboviruses) transmitted by mosquito vectors cause many important emerging or resurging infectious diseases in humans including dengue, chikungunya and Zika. Understanding the co-evolutionary processes among viruses and vectors is essential for the development of novel transmission-blocking strategies. Episomal viral DNA fragments are produced from arboviral RNA upon infection of mosquito cells and adults. Additionally, sequences from insect-specific viruses and arboviruses have been found integrated into mosquito genomes. RESULTS: We used a bioinformatic approach to analyse the presence, abundance, distribution, and transcriptional activity of integrations from 425 non-retroviral viruses, including 133 arboviruses, across the presently available 22 mosquito genome sequences. Large differences in abundance and types of viral integrations were observed in mosquito species from the same region. Viral integrations are unexpectedly abundant in the arboviral vector species Aedes aegypti and Ae. albopictus, in which they are approximately ~10-fold more abundant than in other mosquito species analysed. Additionally, viral integrations are enriched in piRNA clusters of both the Ae. aegypti and Ae. albopictus genomes and, accordingly, they express piRNAs, but not siRNAs. CONCLUSIONS: Differences in the number of viral integrations in the genomes of mosquito species from the same geographic area support the conclusion that integrations of viral sequences is not dependent on viral exposure, but that lineage-specific interactions exist. Viral integrations are abundant in Ae. aegypti and Ae. albopictus, and represent a thus far underappreciated component of their genomes. Additionally, the genome locations of viral integrations and their production of piRNAs indicate a functional link between viral integrations and the piRNA pathway. These results greatly expand the breadth and complexity of small RNA-mediated regulation and suggest a role for viral integrations in antiviral defense in these two mosquito species.

Codon Pair Bias Is a Direct Consequence of Dinucleotide Bias.

Codon pair bias is a remarkably stable characteristic of a species. Although functionally uncharacterized, robust virus attenuation was achieved by recoding of viral proteins using underrepresented codon pairs. Because viruses replicate exclusively inside living cells, we posited that their codon pair preferences reflect those of their host(s). Analysis of many human viruses showed, however, that the encoding of viruses is influenced only marginally by host codon pair preferences. Furthermore, examination of codon pair preferences of vertebrate, insect, and arthropod-borne viruses revealed that the latter do not utilize codon pairs overrepresented in arthropods more frequently than other viruses. We found, however, that codon pair bias is a direct consequence of dinucleotide bias. We conclude that codon pair bias does not play a major role in the encoding of viral proteins and that virus attenuation by codon pair deoptimization has the same molecular underpinnings as attenuation based on an increase in CpG/TpA dinucleotides.

Enveloped virus-like particles as vaccines against pathogenic arboviruses.

Arthropod-borne arboviruses form a continuous threat to human and animal health, but few arboviral vaccines are currently available. Advances in expression technology for complex, enveloped virus-like particles (eVLPs) create new opportunities to develop potent vaccines against pathogenic arboviruses. In this short review, I highlight the successes and challenges in eVLP production for members of the three major arbovirus families: Flaviviridae (e.g., dengue, West Nile, Japanese encephalitis); Bunyaviridae (e.g., Rift Valley fever); and Togaviridae (e.g., chikungunya). The results from pre-clinical testing will be discussed as well as specific constraints to the large-scale manufacture and purification of eVLPs, which are complex assemblies of membranes and viral glycoproteins. Insect cells emerge as ideal substrates for correct arboviral glycoprotein folding and posttranslational modification to yield high quality eVLPs. Furthermore, baculovirus expression in insect cell culture is scalable and has a proven safety record in industrial human and veterinary vaccine manufacturing. In conclusion, eVLPs produced in insect cells using modern biotechnology have a realistic potential to be used in novel vaccines against arboviral diseases.

Exploiting mosquito sugar feeding to detect mosquito-borne pathogens.

Arthropod-borne viruses (arboviruses) represent a global public health problem, with dengue viruses causing millions of infections annually, while emerging arboviruses, such as West Nile, Japanese encephalitis, and chikungunya viruses have dramatically expanded their geographical ranges. Surveillance of arboviruses provides vital data regarding their prevalence and distribution that may be utilized for biosecurity measures and the implementation of disease control strategies. However, current surveillance methods that involve detection of virus in mosquito populations or sero-conversion in vertebrate hosts are laborious, expensive, and logistically problematic. We report a unique arbovirus surveillance system to detect arboviruses that exploits the process whereby mosquitoes expectorate virus in their saliva during sugar feeding. In this system, infected mosquitoes captured by CO(2)-baited updraft box traps are allowed to feed on honey-soaked nucleic acid preservation cards within the trap. The cards are then analyzed for expectorated virus using real-time reverse transcription-PCR. In field trials, this system detected the presence of Ross River and Barmah Forest viruses in multiple traps deployed at two locations in Australia. Viral RNA was preserved for at least seven days on the cards, allowing for long-term placement of traps and continuous collection of data documenting virus presence in mosquito populations. Furthermore no mosquito handling or processing was required and cards were conveniently shipped to the laboratory overnight. The simplicity and efficacy of this approach has the potential to transform current approaches to vector-borne disease surveillance by streamlining the monitoring of pathogens in vector populations.

Application of modified shell vial culture procedure for arbovirus detection.

The isolation of arboviruses from patient's low titer sera can be difficult. Here we compared the detection efficiency of Dengue (DEN), Yellow Fever (YF), Saint Louis Encephalitis (SLE), West Nile (WN), Ilheus (ILH), Group C (GC), Oropouche (ORO), Mayaro (MAY) and Venezuela Encephalitis Equine (VEE) viruses using a Modified Shell Vial Culture (MSVC) protocol to a Standard Cell Culture (SCC) protocol. First the MSVC and SCC protocols were compared using five dilutions for each of the following stock viruses: DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, WN, ILH, GC, ORO, MAY and VEE. Next, patients' original sera from which viruses (DEN-1, DEN-2, DEN-3, YF, GC, ORO, MAY and VEE) had been previously isolated were compare by the two methods using five sera dilutions. In addition, seven sera that were positive for DEN-3 by RT-PCR and negative by SCC were processed by MSVC. The MSVC protocol was consistently 1-2 logs higher virus dilution more sensitive for virus detection than the SCC protocol for all stock Flaviviruses tested (DEN-1, DEN-2, DEN-3, DEN-4, YF, SLE, WN and ILH). MSVC was equal to or one log more sensitive for virus detection than SCC for the stock Bunyaviruses (GC and ORO). For the stock Alphavirus MAY, MSVC was equally or one log more sensitive for virus detection than SCC, while for VEE SCC was equally or one log more sensitive for virus detection than MSVC. MSVC was consistently one to two sera dilutions more sensitive than SCC for the detection of Flaviviruses from patients' sera. Both methods were approximately equally sensitive for the detection of Bunyaviruses from patients' sera and equal or one dilution less sensitive for the detection of Alphaviruses from patients' sera. Additionally, MSVC detected DEN virus in five of seven DEN-3 RT-PCR positive, SCC negative patients' sera.