Molecular Basis for Arbovirus Transmission by Aedes aegypti Mosquitoes.

Mosquitoes are considered the most important vectors for the transmission of pathogens to humans. Aedes aegypti is a unique species, not only by its highly anthropophilic and peridomestic habits but also because it can transmit an important variety of pathogenic viruses. Examples are dengue, yellow fever, chikungunya, Zika, and Mayaro viruses. After ingesting viremic blood, a wide range of mechanisms are activated in the mosquito to counteract viral infection. Nevertheless, these arboviruses possess strategies to overcome barriers in the mosquito and eventually reach the salivary glands to continue the transmission cycle. However, the infection and eventual transmission of arbovirus depends on multiple factors. The current review focuses in detail on the anatomic, physiological, and molecular characteristics of the mosquito A. aegypti that participate in response to a viral infection. In the past decades, the awareness of the importance of this mosquito as a disease vector and its impact on human health was largely recognized. We need to improve our comprehension of molecular mechanisms that determine the outcome of successful virus replication or control of infection for each arbovirus in the vector; this could lead to the design of effective control strategies in the future.

Insect ribosome-rescuer Pelo-Hbs1 complex on sperm surface mediates paternal arbovirus transmission.

Arboviruses can be paternally transmitted by male insects to offspring for long-term persistence, but the mechanism remains largely unknown. Here, we use a model system of a destructive rice reovirus and its leafhopper vector to find that insect ribosome-rescuer Pelo-Hbs1 complex expressed on the sperm surface mediates paternal arbovirus transmission. This occurs through targeting virus-containing tubules constituted by viral nonstructural protein Pns11 to sperm surface via Pns11-Pelo interaction. Tubule assembly is dependent on Hsp70 activity, while Pelo-Hbs1 complex inhibits tubule assembly via suppressing Hsp70 activity. However, virus-activated ubiquitin ligase E3 mediates Pelo ubiquitinated degradation, synergistically causing Hbs1 degradation. Importantly, Pns11 effectively competes with Pelo for binding to E3, thus antagonizing E3-mediated Pelo-Hbs1 degradation. These processes cause a slight reduction of Pelo-Hbs1 complex in infected testes, promoting effective tubule assembly. Our findings provide insight into how insect sperm-specific Pelo-Hbs1 complex is modulated to promote paternal virus transmission without disrupting sperm function.

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.

A role for endosomal proteins in alphavirus dissemination in mosquitoes.

Little is known about endosomal pathway proteins involved in arthropod-borne virus (arbovirus) assembly and cell-to-cell spread in vector mosquitoes. UNC93A and synaptic vesicle-2 (SV2) proteins are involved in intracellular transport in mammals. They show amino acid sequence conservation from mosquitoes to humans, and their transcripts are highly enriched in Aedes aegypti during arbovirus infection. Transient gene silencing of SV2 or UNC93A in mosquitoes infected with the recombinant alphavirus Sindbis MRE16-enhanced green fluorescent protein (SINV; family Togaviridae) resulted in the accumulation of viral positive- and negative-strand RNA, congregation of virus envelope antigen in intracellular networks, and reduced virus dissemination outside of the midgut. Further, UNC93A silencing, but not SV2 silencing, resulted in a 10-fold reduction in viral titres at 4 days post-infection. Together, these data support a role for UNC93A and SV2 in virus assembly or budding. Cis-regulatory elements (CREs) were identified at the 5'-ends of genes from the original data set in which SV2 and UNC93A were identified. Common CREs at the 5'-end genomic regions of a subset of enriched transcripts support the hypothesis that UNC93A transcription may be co-regulated with that of other ion transport and endosomal trafficking proteins.

Systematic identification of autophagy-related proteins in Aedes albopictus.

Autophagy is a conserved cellular process playing a role in maintenance of cellular homeostasis and response to changing nutrient conditions via degradation and recirculation of cellular redundant components. Autophagy-related proteins (Atg) play important function in autophagy pathway. Aedes albopictus mosquito is an effective vector transmitting multiple viruses which cause serious human diseases. Moreover, Aedes albopictus mosquito is becoming a serious threat to human health due to its widening distribution in recent years and thus worth of more research attention. It was reported that autophagy might play a role in viral infection in Aedes mosquito. To better understand the interaction between autophagy and arbovirus infection in mosquito system, it is necessary to identify autophagy pathway in the system. However, autophagy in Aedes albopictus mosquito is still poorly understood so far. We recently identified AaAtg8, the first Atg protein reported in Aedes albopictus mosquito. This work further identified twelve atg genes in Aedes albopictus mosquito. Sequence and phylogenetic analysis of the twelve atg genes were performed. Expression profiles of all the twelve Aaatg genes in different developmental stages and genders of Aedes albopictus mosquito were conducted. Effects of chemicals inhibiting or inducing autophagy on the levels of eight identified AaAtg proteins were examined. The function of two identified AaAtg proteins AaAtg6 and AaAtg16 and their response to arbovirus SINV infection were studied preliminarily. Taken together, this work systematically identified Aedes albopictus atg genes and provided basic information which might help to elucidate the autophagy pathway and the role of autophagy in arbovirus infection in Aedes mosquito system.

Pan-viral protection against arboviruses by activating skin macrophages at the inoculation site.

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The abundance of genetically distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made the development of virus-specific treatments challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical TLR7 agonist suppressed both the local and subsequent systemic course of infection with a variety of arboviruses from the Alphavirus, Flavivirus, and Orthobunyavirus genera. Clinical outcome was improved in mice after infection with a model alphavirus. In the absence of treatment, antiviral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, stimulating the more populous skin-resident macrophages with a TLR7 agonist elicited protective responses in key cellular targets of virus that otherwise proficiently replicated virus. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have identified a putative new strategy for limiting disease after infection with a variety of genetically distinct arboviruses.

Metabolomic Insights into Human Arboviral Infections: Dengue, Chikungunya, and Zika Viruses.

The global burden of arboviral diseases and the limited success in controlling them calls for innovative methods to understand arbovirus infections. Metabolomics has been applied to detect alterations in host physiology during infection. This approach relies on mass spectrometry or nuclear magnetic resonance spectroscopy to evaluate how perturbations in biological systems alter metabolic pathways, allowing for differentiation of closely related conditions. Because viruses heavily depend on host resources and pathways, they present unique challenges for characterizing metabolic changes. Here, we review the literature on metabolomics of arboviruses and focus on the interpretation of identified molecular features. Metabolomics has revealed biomarkers that differentiate disease states and outcomes, and has shown similarities in metabolic alterations caused by different viruses (e.g., lipid metabolism). Researchers investigating such metabolomic alterations aim to better understand host⁻virus dynamics, identify diagnostically useful molecular features, discern perturbed pathways for therapeutics, and guide further biochemical research. This review focuses on lessons derived from metabolomics studies on samples from arbovirus-infected humans.

Multiplex microsphere immunoassays for the detection of IgM and IgG to arboviral diseases.

Serodiagnosis of arthropod-borne viruses (arboviruses) at the Division of Vector-Borne Diseases, CDC, employs a combination of individual enzyme-linked immunosorbent assays and microsphere immunoassays (MIAs) to test for IgM and IgG, followed by confirmatory plaque-reduction neutralization tests. Based upon the geographic origin of a sample, it may be tested concurrently for multiple arboviruses, which can be a cumbersome task. The advent of multiplexing represents an opportunity to streamline these types of assays; however, because serologic cross-reactivity of the arboviral antigens often confounds results, it is of interest to employ data analysis methods that address this issue. Here, we constructed 13-virus multiplexed IgM and IgG MIAs that included internal and external controls, based upon the Luminex platform. Results from samples tested using these methods were analyzed using 8 different statistical schemes to identify the best way to classify the data. Geographic batteries were also devised to serve as a more practical diagnostic format, and further samples were tested using the abbreviated multiplexes. Comparative error rates for the classification schemes identified a specific boosting method based on logistic regression "Logitboost" as the classification method of choice. When the data from all samples tested were combined into one set, error rates from the multiplex IgM and IgG MIAs were <5% for all geographic batteries. This work represents both the most comprehensive, validated multiplexing method for arboviruses to date, and also the most systematic attempt to determine the most useful classification method for use with these types of serologic tests.

Distribution of virus structural proteins and protein-protein interactions in plasma membrane of baby hamster kidney cells infected with Sindbis or vesicular stomatitis virus.

The plasma membrane of baby hamster kidney (BHK-21) cells infected with either Sindbis or vesicular stomatitis virus was isolated by a technique involving the ingestion of latex beads by the cells. Plasma membrane isolated from Sindbis virus-infected cells contained only one (E1) of the three (E1, E2, and C) structural proteins of this virus. When the latex beads were pretreated with either polylysine or DEAE-dextran, plasma membrane obtained from Sindbis virus-infected cells contained all three structural proteins and PE2, a precursor to one of the structural proteins. In pulse-chase radiolabeling experiments with Sindbis virus-infected cells, it was possible to follow the appearance of the precursor protein (PE2) i the plasma membrane and its eventual conversion to E2. The appearance of Sindbis virus membrane proteins PE2 and E1 in the purified plasma membrane was not affected by the drug tunicamycin, an inhibitor of glycosylation. These experiments imply the following: (i) Cleavage of the Sindbis virus precursor polypeptide PE2 to E2 is not a prerequisite for its transport to the cell plasma membrane; (ii) transport of virus membrane proteins to the cell surface does not depend on glycosylation; and (iii) although all Sindbis virus structural proteins are associated with the plasma membrane, a generally accepted pairing of PE2-E1 or E2-E1 in the plasma membrane either does not exist or, if it does exist, involves a very weak interaction. The procedures used in this study also resulted in the successful isolation of plasma membrane from vesicular stomatitis virus-infected cells containing the glycoprotein, the matrix protein, and the nucleocapsid protein, a result that suggests that these proteins are located on the media side of baby hamster kidney cells grown in monolayer.

Receptor alterations associated with spinal motoneuron degeneration in bovine Akabane disease.

Akabane disease in cattle is characterized by congenital abnormalities including arthrogryposis, which is characterized by a depletion of spinal ventral horn motoneurons, a loss of axons, and depletion of myelin in the lateral and ventral tracts. These neuropathological changes produced major reductions (70-80%) in the density of muscarinic cholinergic, glycine/strychnine, and central-type benzodiazepine receptors in the ventral horn motor nuclei. The density of peripheral-type benzodiazepine receptors and adenosine A1 receptors was dramatically increased (250-300%) in the lateral and ventral spinal columns, reflecting the proliferation of glial cells. Bovine Akabane disease represents a useful model for assessing the processes and consequences of neuronal degeneration and demyelination and has implications for human diseases such as amyotrophic lateral sclerosis.

Effect of interferon on togavirus and arenavirus infections of animals.

The sensitivity of togaviruses to the effects of IF appears to permit successful exploitation of IF inducers, whereas the relative insensitivity of arenaviruses to IF in vitro appears to correlate with the complete lack of success of poly (ICLC) therapy in the monkey model. Noteworthy is the successful use of poly (ICLC) against fatal encephalitis caused by JE virus in monkeys. Additional studies appear to be warranted to characterize further the role of IF and/or IF inducers in other model infections prior to studies in man.

The role of complement in viral infections. II. the clearance of Sindbis virus from the bloodstream and central nervous system of mice depleted of complement.

The following studies were performed to investigate the mechanism(s) by which the complement system limits Sindbis virus infection in the central nervous system of mice. After the intracerebral inoculation of Sindbis virus, no differences in mortality or viral growth in the central nervous system were observed between normal mice and mice depleted of complement by treatment with cobra venom factor. In addition, animals that had been inoculated subcutaneously with Sindbis virus and depleted of complement after the viremic phase had ended did not show any differences in mortality or viral growth in the central nervous system. In contrast, it was found that after the intracardiac inoculation of virus, complement-depleted mice demonstrated a defect in the clearance of infectious virus from the blood. These studies suggest that the increased growth of virus in the brains of complement-depleted mice after the subcutaneous inoculation of Sindbis virus is due to a defect in clearance of infectious virus from the bloodstream rather than to a primary defect within the central nervous system.