Infection with Zika virus (ZIKV) is of growing concern since infection is associated with the development of congenital neurological disease. Quantitative reverse transcription PCR (qRT-PCR) has been the standard for ZIKV detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper testing. Studies have suggested that ZIKV detection in urine is more sensitive and has a longer window of detection compared to serum and saliva. The objective of this study was to develop a urine diagnostic test that could be completed in under 30 minutes. Urine samples spiked with ZIKV or dengue virus were tested using RT-LAMP as well as by conventional quantitative qRT-PCR. These techniques were then validated using crude lysates made from ZIKV infected mosquitoes in addition to urine and serum samples from ZIKV infected patients. RT-LAMP specifically detected ZIKV in urine and serum for ZIKV infected patients and crude mosquito lysates. This test was performed in under 30 minutes and did not require RNA extraction from urine nor mosquitos. This approach could be used for monitoring of exposed individuals, especially pregnant women, couples wanting to conceive, or individuals with suspicious symptoms as well as surveillance of mosquito populations.

In higher eukaryotic cells, mitochondria are essential subcellular organelles for energy production, cell signaling, and the biosynthesis of biomolecules. The mitochondrial DNA (mtDNA) genome is indispensable for mitochondrial function because it encodes protein subunits of the electron transport chain and a full set of transfer and ribosomal RNAs. MtDNA degradation has emerged as an essential quality control measure to maintain mtDNA and to cope with mtDNA damage resulting from endogenous and environmental factors. Among all types of DNA damage known, abasic (AP) sites, sourced from base excision repair and spontaneous base loss, are the most abundant endogenous DNA lesions in cells. In mitochondria, AP sites trigger rapid DNA loss; however, the mechanism and molecular factors involved in the process remain elusive. Herein, we demonstrate that the stability of AP sites is reduced dramatically upon binding to a major mtDNA packaging protein, mitochondrial transcription factor A (TFAM). The half-life of AP lesions within TFAM–DNA complexes is 2 to 3 orders of magnitude shorter than that in free DNA, depending on their position. The TFAM-catalyzed AP-DNA destabilization occurs with nonspecific DNA or mitochondrial light-strand promoter sequence, yielding DNA single-strand breaks and DNA–TFAM cross-links. TFAM–DNA cross-link intermediates prior to the strand scission were also observed upon treating AP-DNA with mitochondrial extracts of human cells. In situ trapping of the reaction intermediates (DNA–TFAM cross-links) revealed that the reaction proceeds via Schiff base chemistry facilitated by lysine residues. Collectively, our data suggest a novel role of TFAM in facilitating the turnover of abasic DNA.

Aedes aegypti is the primary vector of a number of viruses pathogenic to humans including dengue virus (DENV). DENV infection leads to widespread transcriptomic and proteomic alterations in mosquito cells. Here we identified alterations to the mosquito cell secretome during DENV infection by performing liquid chromatography tandem mass spectrometry. We found that an extracellular fragment of low-density lipoprotein receptor-related protein 1 (LRP-1) was present during infection. Previous literature suggests that LRP-1 regulates cholesterol homeostasis. Therefore, we hypothesized that DENV modifies LRP-1 protein expression to maintain host-derived intracellular cholesterol, which would facilitate virus replication within membrane-associated replication compartments. Accordingly, stimuli that are present during flavivirus infection reduced LRP-1 protein expression. We also found that dsRNA knockdown of LRP-1 increased intracellular cholesterol and DENV viral RNA. Further, depletion of intracellular lipids reduced infection. Together, these data suggest that DENV reduces LRP-1 protein expression, possibly through regulated intramembrane proteolysis (RIP), to increase intracellular cholesterol and facilitate replication in Ae. aegypti .

Zika virus (ZIKV) is a rapidly emerging flavivirus that has been associated with a number of congenital neurological manifestations. Here, we show that ZIKV replicated efficiently in mouse neural stem cells (mNSCs). ZIKV infection caused a cytopathic effect without affecting cell viability, yet led to a significant decrease in the number of proteins secreted into mNSC supernatants. A gene expression array of neural stem cell progenitor and differentiation markers suggested that infection reduced the number of neuronal and oligodendrocyte progenitors while increasing the number of astrocyte progenitors. Infection in astrocytes increased transcription of key genes involved in the antiviral response. These data provide molecular and cellular evidence that ZIKV significantly alters neural development in the vertebrate host and that astrocyte differentiation may be a protective response that limits neuropathogenesis.

Aedes aegypti is the primary vector of a number of human pathogens including dengue virus (DENV) and Zika virus (ZIKV). Ae. aegypti acquires these viruses during the processing of bloodmeals obtained from an infected vertebrate host. Vertebrate blood contains a number of factors that have the potential to modify virus acquisition in the mosquito. Interestingly, low density lipopolyprotein (LDL) levels are decreased during severe DENV infection. Accordingly, we hypothesized that LDL is a modifiable factor that can influence flavivirus acquisition in the mosquito. We found that LDL is endocytosed by Ae. aegypti cells in a dynamin-dependent manner. LDL is also endocytosed by midgut epithelial cells and accumulates at the luminal midgut epithelium during bloodmeal digestion. Importantly, pretreatment with LDL, but not high density lipopolyprotein (HDL), significantly inhibited both DENV and ZIKV infection in vitro, and LDL inhibited ZIKV infection in vivo. This study identifies human LDL or 'bad cholesterol' as a modifiable factor that can inhibit flavivirus acquisition in Ae. aegypti. Identification of modifiable blood factors and critical cellular interactions that mediate pathogen acquisition may lead to novel strategies to disrupt the transmission cycle of vector-borne diseases.

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Arboviruses are a large group of viruses that are transmitted by arthropods including ticks and mosquitoes. The global diversity of arboviruses is unknown; however, theoretical studies have estimated that over 2,000 mosquito-borne flaviviruses may exist. An increasing number of flaviviruses can only infect insect cells. We hypothesize that insect-specific flaviviruses (ISFVs) represent model genetic precursors to pathogenic flaviviruses, although the genetic mechanisms required for adaptation to vertebrate hosts are unclear. In this study, we determined that Kamiti River virus (KRV) infection was inhibited by innate immunity pathways in vertebrate cells. KRV infection of IRF3,5,7(-/-) mouse embryonic fibroblasts led to low levels of viral protein production and shedding of infectious progeny. These data suggest that ISFVs cannot evade vertebrate innate immune pathways. Identifying cellular pathways and genetic changes that are required for adaptation of arthropod-specific arboviruses to vertebrate hosts is critical to understanding emerging infectious disease.

PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase (AEP) superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn 2+ , a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas, in the presence of Mg 2+ , the chemical step becomes rate-limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn 2+ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors, and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.