Jeffrey Grabowski

Foundation for Advanced Education in the Sciences at the NIH

Tick‐borne viruses cause thousands of cases of disease worldwide every year. Specific countermeasures to many tick‐borne viruses are not commercially available. Very little is known regarding tick‐virus interactions and increasing this knowledge can lead to potential targets for countermeasure development. Virus infection of ex vivo organ cultures...

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 unde...

The unfolded protein response (UPR) maintains protein-folding homeostasis in the endoplasmic reticulum (ER) and has been implicated as both beneficial and detrimental to flavivirus infection. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a sensor of the UPR, is commonly associated with antiviral effects during mosquito-borne flav...

Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, ho...

Powassan virus (POWV) is the only tick-borne flavivirus (TBFV) known to circulate in North America. Although there are relatively few documented cases of POWV disease, investigations into POWV are justified due to the increasing incidence of infection and significant case fatality rate associated with this virus. To better describe the molecular bi...

Tick-borne flaviviruses (TBFVs) are responsible for more than 15,000 human disease cases each year, and Powassan virus lineage 2 (POWV-L2) deer tick virus has been a reemerging threat in North America over the past 20 years. Rapid transmission of TBFVs in particular emphasizes the importance of preventing tick bites, the difficulty in developing co...

Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) poses new challenges for controlling virus outbreaks and developing live-attenuated vaccines. To elucidate routes of ZIKV dissemination in the MRT, we here generate microRNA-targeted ZIKV clones that lose the infectivity for (1) the cells inside seminifero...

Each year there are more than 15 000 cases of human disease caused by infections with tick-borne viruses (TBVs). These illnesses occur worldwide and can range from very mild illness to severe encephalitis and hemorrhagic fever. Although TBVs are currently identified as neglected vector-borne pathogens and receive less attention than mosquito-borne...

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 environ...

Ae. aegypti sample preparation for LC-MS analysis. Flow chart shows timeline, numbers of samples and procedures used for mosquito rearing, infection, sample collection and sample processing. (TIF)

4HPR treatment resulted in increased accumulations of both Cer and DHCer and inhibited DENV replication but did not alter the Cer/DHCer ratios. (A) and (B) Aag2 cells were pre-treated with 3.75 μM of 4HPR or DMSO, a vehicle control. At 24 h post treatment, cells were infected with DENV at MOI of 0.3. Fresh medium with 4HPR or DMSO was replaced at 1...

MRM profiling of additional SPs in Aag2 cells after DEGS-KD By RNAi. Abundance of (A) sphingosine (d18:1), sphingosine-1-phosphate (d18:1-P) and sphinganine (d18:0) and (B) sphingomyelins upon DEGS-KD was compared to GFP-KD control. Student’s t-test was applied for statistical analysis and none of these metabolites had differential abundance upon D...

MRM table for data acquisition of Cer and DHCer (according to Merrill et al. 2005 [124]) and 16 carbon sphingoid-backbone Cer and DHCer (modified from Merrill et al., 2005). (DOCX)

MRM profiling of SPs in Aag2 cells during DENV infection. DENV infected (MOI of 3) or mock infected Aag2 cells were harvested at 24 hpi and processed for SP profiling by MRM (N = 3). (A, lower panel) Cer(d18:1/xx:x) and DHCer(d18:0/xx:x) with 18-carbon long chain sphingoid bases, and (B, lower panel) Cer(d16:1/xx:x) and DHCer(16:0/xx:x) with 16-car...

Gene-specific primers for dsRNA. (DOCX)

Primers for detecting gene expression levels following RNAi knockdown. (DOCX)

Select metabolites from mosquito midguts that show differential abundance following DENV infection. Abundance of metabolites detected in DENV-infected and uninfected midguts was compared. Frist tab lists the molecules that were putatively identifiable and second tab lists the molecules were unidentifiable. The following information is provided for...

Multiple reaction monitoring table for data acquisition of free sphingoid bases and 1-phosphates (according to Merrill et al., 2005 [124]). (DOCX)

MRM table for data acquisition of sphingomyelins (according to Merrill et al., 2005 [124]). (DOCX)

Comparative analysis of fatty acyls in mosquito midguts following DENV infection. Average abundance of fatty acyl molecule in DENV infected midguts was compared with uninfected midguts and represented as log2 fold change. Each row shows a different fatty acyl molecule, grouped based on the classification of molecular structure. Columns represent 3,...

Analysis of free sphingoid bases and 1-phosphate species, analysis of ceramide (16 and 18 carbon sphingoid-backbone) species, analysis of sphingomyelin species. (DOCX)

Tick-borne flaviviruses (TBFs) affect human health globally. Human vaccines provide protection against some TBFs, and antivirals are available, yet TBF-specific control strategies are limited. Advances in genomics offer hope to understand the viral complement transmitted by ticks, and to develop disruptive, data-driven technologies for virus detect...

Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus...

Flaviviruses have an intimate relationship with their host cells, utilizing host proteins during replication. Much of viral genome replication and virion assembly occurs on and within the endoplasmic reticulum (ER). As a cellular protein folding hub, the ER provides an ideal environment for flaviviruses to replicate. Flaviviruses can interact with...

Background: Tick-borne flaviviruses (TBFs) cause thousands of human cases of encephalitis worldwide each year, with some TBF infections progressing to hemorrhagic fever. TBFs are of medical and veterinary importance and strategies to reduce flavivirus transmission by the tick vector may have significant application. Analyses of the proteome of ISE...

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-tr...

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-tr...

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 a...

ISE6 cell viability, growth, and mortality following LGTV infection. Alteration of ISE6 cell viability (A) at 12–24, 24–36, 36–48 hours post infection are shown in parallel with cell growth/population numbers (B) at 24, 36, 48 hours post infection, and cell mortality percentage (C) at 12, 24, 36, 48 hours post infection with and without LGTV (mock-...

I. scapularis proteins with increased expression following LGTV-infection and UV-LGTV treatment. As mentioned in S1A Fig, four groups of categorized proteins were identified: ISE6 ortholog proteins, ISE6 proteins with no orthology, ISE6 ortholog proteins with no mapped I. scapularis cellular pathways, and ISE6 ortholog proteins with mapped cellular...

Summary of analyses used to identify proteins from LGTV-infected and UV-LGTV-treated ISE6 cell samples. (DOCX)

Pathways populated with ISE6 ortholog proteins following LGTV-infection and UV-LGTV treatment. (XLSX)

ISE6 protein orthology and cellular function distribution of proteins found in KEGG pathways and modules. (A) I. scapularis ISE6 proteins with KEGG-mapped orthologs (or KEGG orthology [KO]) help to identify cellular pathways in I. scapularis (genome.jp/kegg/ko). To be identified in a KEGG pathway, KO is required. ISE6 proteins with KO and not ident...

Summary of differentially-expressed ISE6 proteins without identified pathways. Expression of ISE6 proteins with (A) or without (B) orthology and no identified pathways. Refer to S2 Table for more specifics on the proteins. Red dotted line denotes differentially-expressed proteins in LGTV-infected ISE6 cells compared to UV-LGTV-treated ISE6 cells (n...

Number of ISE6 proteins corresponding to orthologous proteins identified in proteomic analyses of flavivirus-host systems. Corresponding percentages correspond to the number of tick ISE6 orthologs identified with orthologs identified in: α S5 Fig, S7 Fig, and S11 Fig of Khadka et al. [56]; μS2 Table of Tchankouo-Nguetcheu et al. [28]; βTables 1 and...

Summary of proteomic analysis of LGTV-infected and UV-LGTV-treated I. scapularis ISE6 cells. After whole cell sample harvest of treated ISE6 cells, cell pellet samples were subject to lipid removal, protein precipitation, peptide denaturation, and tryptic digest of peptides. Samples were prepared for the separation phase (nano LC) by injection, usi...

486 significant, I. scapularis ISE6 proteins identified. The total number of I. scapularis ISE6 proteins is based off of ≥1 peptide identification and ≥1 statistical analysis (p < 0.05) identification (four total analyses). From S1 Table, the filter process in detail is listed and Fig 2 is a pattern representation including the 486 proteins listed...

ISE6 proteins putatively associated with glutaminolysis. (DOCX)

Supplementary Figures 1-25, Supplementary Tables 1-38, Supplementary Note 1, Supplementary Methods and Supplementary References

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 t...

Aedes aegypti mosquitoes are the primary vectors transmitting dengue virus (DENV), one of the most aggressive re-emerging pathogens worldwide causing more than 390 million infections per year. The spread of the virus is greatly dependent upon successful replication within both the human host and mosquito vector. Much effort has been placed in under...

The black-legged tick (Ixodes scapularis) is the primary vector of Borrelia bugdorferi, the bacteria that causes Lyme Disease. The nymphal stage of the tick's life cycle plays a large role in the transmission of Lyme Disease. Suitable nymph microhabitat includes lower vegetation layers, upper layers of the soil column, and leaf litter, which mainta...