Characterization of flavivirus infection in salivary gland cultures from male Ixodes scapularis ticks.

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.

Early mitochondrial dysfunction leads to altered redox chemistry underlying pathogenesis of TPI deficiency.

Triose phosphate isomerase (TPI) is responsible for the interconversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate in glycolysis. Point mutations in this gene are associated with a glycolytic enzymopathy called TPI deficiency. This study utilizes a Drosophila melanogaster model of TPI deficiency; TPI(sugarkill) is a mutant allele with a missense mutation (M80T) that causes phenotypes similar to human TPI deficiency. In this study, the redox status of TPI(sugarkill) flies was examined and manipulated to provide insight into the pathogenesis of this disease. Our data show that TPI(sugarkill) animals exhibit higher levels of the oxidized forms of NAD(+), NADP(+) and glutathione in an age-dependent manner. Additionally, we demonstrate that mitochondrial redox state is significantly more oxidized in TPI(sugarkill) animals. We hypothesized that TPI(sugarkill) animals may be more sensitive to oxidative stress and that this may underlie the progressive nature of disease pathogenesis. The effect of oxidizing and reducing stressors on behavioral phenotypes of the TPI(sugarkill) animals was tested. As predicted, oxidative stress worsened these phenotypes. Importantly, we discovered that reducing stress improved the behavioral and longevity phenotypes of the mutant organism without having an effect on TPI(sugarkill) protein levels. Overall, these data suggest that reduced activity of TPI leads to an oxidized redox state in these mutants and that the alleviation of this stress using reducing compounds can improve the mutant phenotypes.