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Infected Ixodes scapularis (black-legged tick) transmit a host of serious pathogens via their bites, including Borrelia burgdorferi, Babesia microti, and tick-borne flaviviruses (TBFVs), such as Powassan virus (POWV). Although the role of female I. scapularis ticks in disease transmission is well characterized, the role of male ticks is poorly understood. Because the pathogens are delivered in tick saliva, we studied the capacity of male salivary glands (SGs) to support virus replication. Ex vivo cultures of SGs from unfed male I. scapularis were viable for more than a week and maintained the characteristic tissue architecture of lobular ducts and acini. When SG cultures were infected with the TBFVs Langat virus (LGTV) or POWV lineage II (deer tick virus), the production of infectious virus was demonstrated. Using a green fluorescent protein-tagged LGTV and confocal microscopy, we demonstrated LGTV infection within SG acinus types II and III. The presence of LGTV in the acini and lobular ducts of the cultures was also shown via immunohistochemistry. Furthermore, the identification by in situ hybridization of both positive and negative strand LGTV RNA confirmed that the virus was indeed replicating. Finally, transmission electron microscopy of infected SGs revealed virus particles packaged in vesicles or vacuoles adjacent to acinar lumina. These studies support the concept that SGs of male I. scapularis ticks support replication of TBFVs and may play a role in virus transmission, and further refine a useful model system for developing countermeasures against this important group of pathogens. Author summary: Powassan disease has greatly increased in frequency since its discovery in Powassan, Ontario in 1958. Powassan virus (lineage I; POWV) and Powassan virus lineage II (deer tick virus; DTV) are endemic to North America and there were 133 reported cases between 2009 and 2018, the majority since 2016. Nymphal and adult Ixodes scapularis ticks are thought to be the primary vectors of POWV/DTV to humans. However, little is known regarding DTV infection of male Ixodes ticks or their potential as vectors. In this study we characterized LGTV, a model tick-borne flavivirus, and DTV infection and propagation in male I. scapularis salivary gland cultures using an ex vivo organ culture system. This work provides insight into potential flavivirus transmission by the male I. scapularis tick. [ABSTRACT FROM AUTHOR]

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes. [ABSTRACT FROM AUTHOR]

Background: Ticks (Family Ixodidae) transmit a variety of disease causing agents to humans and animals. The tick-borne flaviviruses (TBFs; family Flaviviridae) are a complex of viruses, many of which cause encephalitis and hemorrhagic fever, and represent global threats to human health and biosecurity. Pathogenesis has been well studied in human and animal disease models. Equivalent analyses of tick-flavivirus interactions are limited and represent an area of study that could reveal novel approaches for TBF control. Methodology/Principal Findings: High resolution LC-MS/MS was used to analyze the proteome of Ixodes scapularis (Lyme disease tick) embryonic ISE6 cells following infection with Langat virus (LGTV) and identify proteins associated with viral infection and replication. Maximal LGTV infection of cells and determination of peak release of infectious virus, was observed at 36 hours post infection (hpi). Proteins were extracted from ISE6 cells treated with LGTV and non-infectious (UV inactivated) LGTV at 36 hpi and analyzed by mass spectrometry. The Omics Discovery Pipeline (ODP) identified thousands of MS peaks. Protein homology searches against the I. scapularis IscaW1 genome assembly identified a total of 486 proteins that were subsequently assigned to putative functional pathways using searches against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. 266 proteins were differentially expressed following LGTV infection relative to non-infected (mock) cells. Of these, 68 proteins exhibited increased expression and 198 proteins had decreased expression. The majority of the former were classified in the KEGG pathways: “translation”, “amino acid metabolism”, and “protein folding/sorting/degradation”. Finally, Trichostatin A and Oligomycin A increased and decreased LGTV replication in vitro in ISE6 cells, respectively. Conclusions/Significance: Proteomic analyses revealed ISE6 proteins that were differentially expressed at the peak of LGTV replication. Proteins with increased expression following infection were associated with cellular metabolic pathways and glutaminolysis. In vitro assays using small molecules implicate malate dehydrogenase (MDH2), the citrate cycle, cellular acetylation, and electron transport chain processes in viral replication. Proteins were identified that may be required for TBF infection of ISE6 cells. These proteins are candidates for functional studies and targets for the development of transmission-blocking vaccines and drugs. [ABSTRACT FROM AUTHOR]

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption. [ABSTRACT FROM AUTHOR]

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption.