Comparative Pathogenesis of Two Lineages of Powassan Virus Reveals Distinct Clinical Outcome, Neuropathology, and Inflammation.

Tick-borne flaviviruses (TBFV) can cause severe neuroinvasive disease which may result in death or long-term neurological deficit in over 50% of survivors. Multiple mechanisms for invasion of the central nervous system (CNS) by flaviviruses have been proposed including axonal transport, transcytosis, endothelial infection, and Trojan horse routes. Flaviviruses may utilize different or multiple mechanisms of neuroinvasion depending on the specific virus, infection site, and host variability. In this work we have shown that the infection of BALB/cJ mice with either Powassan virus lineage I (Powassan virus) or lineage II (deer tick virus) results in distinct spatial tropism of infection in the CNS which correlates with unique clinical presentations for each lineage. Comparative transcriptomics of infected brains demonstrates the activation of different immune pathways and downstream host responses. Ultimately, the comparative pathology and transcriptomics are congruent with different clinical signs in a murine model. These results suggest that the different disease presentations occur in clinical cases due to the inherent differences in the two lineages of Powassan virus.

Evaluation of right ventricular strain in two separate cohorts with precapillary pulmonary hypertension.

Evaluation for right ventricular (RV) dysfunction is an important part of risk assessment in care of patients with pulmonary hypertension (PH) as it is associated with morbidity and mortality. Echocardiography provides a widely available and acceptable method to assess RV function. RV global longitudinal strain (RVGLS), a measure of longitudinal shortening of RV deep muscle fibers obtained by two-dimensional echocardiography, was previously shown to predict short-term mortality in patients with PH. The purpose of the current study was to assess the performance of RVGLS in predicting 1-year outcomes in PH. We retrospectively identified 83 subjects with precapillary PH and then enrolled 50 consecutive prevalent pulmonary arterial hypertension (PAH) subjects into a prospective validation cohort. Death as well as combined morbidity and mortality events at 1 year were assessed as outcomes. In the retrospective cohort, 84% of patients had PAH and the overall 1-year mortality rate was 16%. Less negative RVGLS was marginally better than tricuspid annular plane systolic excursion (TAPSE) as a predictor for death. However, in the prospective cohort, 1-year mortality was only 2%, and RVGLS was not predictive of death or a combined morbidity and mortality outcome. This study supports that RV strain and TAPSE have similar 1-year outcome predictions but highlights that low TAPSE or less negative RV strain measures are often false-positive in a cohort with low baseline mortality risk. While RV failure is considered the final common pathway for disease progression in PAH, echocardiographic measures of RV function may be less informative of risk in serial follow-up of treated PAH patients.

Powassan Virus Lineage I in Field-Collected Dermacentor variabilis Ticks, New York, USA.

Powassan virus is a tickborne flavivirus that can cause lethal or debilitating neurologic illness. It is canonically transmitted by Ixodes spp. ticks but might spill over to sympatric Dermacentor species. We detected Powassan virus lineage I from a pool of field-collected D. variabilis ticks in New York, USA.

Human attachment site preferences of ticks parasitizing in New York.

Ticks transmit several arthropod-borne pathogens in New York State. The primary human-biting ticks in this region are Ixodes scapularis, Amblyomma americanum, and Dermacentor variabilis. Body regions where tick bites human vary depending on the tick species and life stage, and clothing worn by the host. A community tick submission system was used to acquire information about bite-site location prior to pathogen testing to understand species and life stage-specific body-segment preferences. These data resulted in the identification of species-specific preferences for location, with D. variabilis preferentially biting the head and neck and A. americanum preferring the thighs, groin, and abdomen. Ixodes scapularis was found across the body, although it showed a significant life stage difference with adults preferring the head, midsection, and groin, while nymphs/larvae preferred the extremities. Infection with Borrelia burgdorferi resulted in a significant change in attachment site. This provides an assessment of which body region ticks of the most common species in New York are likely to be found.

Pulmonary hypertension mortality trends in United States 1999-2019.

PURPOSE: Pulmonary hypertension (PH) is a heterogenous, often progressive disorder leading to right heart failure and death. Previous analyses show stable PH mortality rates from 1980 to 2001 but increasing from 2001 to 2010 especially among women and non-Hispanic (NH) Black. This study seeks to identify recent trends in PH mortality in the United States from 1999 to 2019. METHODS: Mortality rates among individuals more than or equal to 15 years of age were obtained from the Centers for Disease Control and Prevention's (CDC) Wide-Ranging Online Data for Epidemiology Research (WONDER) database. ICD-10 codes were used to identify individuals with PH. RESULTS: Between 1999 and 2019, PH was included as a cause on 429,105 recorded deaths. The average age-adjusted PH mortality rate was 7.9 per 100,000 individuals and increased by 1.9% per year. Higher age-adjusted mortality rates were experienced by females and NH Black persons. The crude mortality rate was 105.4 per 100,000 among those decedents 85 or older. From 1999 to 2019, mortality in PH and left heart disease co-occurrence increased at nearly double the annual rate of the overall PH group. CONCLUSIONS: Despite therapeutic advances for selected PH subgroups, the overall age-adjusted PH mortality rate increased significantly from 1999 to 2019 and previously reported racial disparities have persisted. These findings emphasize the need for additional study to improve outcomes in PH.

Infection with Borrelia burgdorferi Increases the Replication and Dissemination of Coinfecting Powassan Virus in Ixodes scapularis Ticks.

Powassan virus (POWV) is a tick-borne neuroinvasive flavivirus endemic to North America. It is generally transmitted by the tick, Ixodes scapularis. This species also transmits Borrelia burgdorferi, the causative agent of Lyme disease. Infection with B. burgdorferi can result in arthritis, carditis, and neuroborreliosis. These pathogens experience sylvatic overlap. To determine the risk of human exposure to coinfected ticks, the interactions between POWV and B. burgdorferi are assessed in laboratory-infected I. scapularis. Adult male and female I. scapularis ticks are orally inoculated with either both pathogens, POWV only, B. burgdorferi only, or uninfected media. After twenty-one days, the ticks are dissected, and RNA is extracted from their midguts and salivary glands. In infected midguts, the quantity of POWV in coinfected ticks was elevated compared to those with only POWV. In addition, the salivary glands of ticks with infected midguts had increased POWV dissemination to those with only POWV. RNA sequencing is performed to identify the potential mechanism for this pattern, which varies between the organs. Ixodes scapularis ticks are found to be capable of harboring both POWV and B. burgdorferi with a benefit to POWV replication and dissemination.

Established Populations of Rickettsia parkeri-Infected Amblyomma maculatum Ticks in New York City, New York, USA.

Objectives: We sought to determine the habitat associations and pathogen status of Amblyomma maculatum ticks in New York City (NYC), New York, USA, a newly expanded portion of their range. Methods: We collected 88 ticks from two NYC parks on Staten Island, one of the five boroughs of NYC, and compared our findings with similar habitat in Brooklyn, New York during the same time period (April 30-September 1). We tested 76 for pathogens. Results: We found adult and immature ticks in native and invasive grasses at Freshkills and Brookfield parks on Staten Island. No A. maculatum ticks were found in Brooklyn. 52.6% of ticks tested were infected with Rickettsia parkeri-the etiological agent of R. parkeri rickettsiosis. Conclusions: This high rate of R. parkeri in a dense urban center is of concern to the medical community, who should be aware of this species' presence and the symptoms of R. parkeri rickettsiosis.

Community engaged tick surveillance and tickMAP as a public health tool to track the emergence of ticks and tick-borne diseases in New York.

A community engaged passive surveillance program was utilized to acquire ticks and associated information throughout New York state. Ticks were speciated and screened for several tick-borne pathogens. Of these ticks, only I. scapularis was commonly infected with pathogens of human relevance, including B. burgdorferi, B. miyamotoi, A. phagocytophilum, B. microti, and Powassan virus. In addition, the geographic and temporal distribution of tick species and pathogens was determined. This enabled the construction of a powerful visual analytical mapping tool, tickMAP to track the emergence of ticks and tick-borne pathogens in real-time. The public can use this tool to identify hot-spots of disease emergence, clinicians for supportive evidence during differential diagnosis, and researchers to better understand factors influencing the emergence of ticks and tick-borne diseases in New York. Overall, we have created a community-engaged tick surveillance program and an interactive visual analytical tickMAP that other regions could emulate to provide real-time tracking and an early warning for the emergence of tick-borne diseases.

Tick-virus interactions: Current understanding and future perspectives.

Ticks are the primary vector of arboviruses in temperate climates worldwide. They are both the vector of these pathogens to humans and an integral component of the viral sylvatic cycle. Understanding the tick-pathogen interaction provides information about the natural maintenance of these pathogens and informs the development of countermeasures against human infection. In this review, we discuss currently available information on tick-viral interactions within the broader scope of general tick immunology. While the tick immune response to several pathogens has been studied extensively, minimal work centres on responses to viral infection. This is largely due to the high pathogenicity of tick-borne viruses; this necessitates high-containment laboratories or low-pathogenicity substitute viruses. This has biased most research towards tick-borne flaviviruses. More work is required to fully understand the role of tick-virus interaction in sylvatic cycling and transmission of diverse tick-borne viruses.

Microbial interactions in the mosquito gut determine Serratia colonization and blood-feeding propensity.

How microbe-microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that, Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however, one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behavior; Serratia exposure to Ae. aegypti disrupted their feeding behavior, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host-microbe interactions and provides evidence that microbial interactions influence mosquito behavior.

Influence of Body Weight and Diabetes Mellitus in Patients With Pulmonary Hypertension.

Pulmonary hypertension (PH) is a complex condition that arises due to pulmonary vascular disease, heart disease, lung disease, chronic thromboembolism, or several rare causes. Regardless of underlying cause, PH increases mortality, yet there are no directed treatments for the most common forms of PH due to left heart or lung disease. Because metabolic factors have been implicated in the pathogenesis of PH, we used a large administrative cohort to assess diabetes and weight, potentially modifiable risk factors, on PH outcome. We analyzed 110,495 veterans diagnosed with PH from January 1, 2003 to September 30, 2015 in the Veterans Health Affairs system. Veterans with PH survived an average of 3.88 [IQR 3.85, 3.92] years after PH diagnosis. Diabetes occurred in 36% and increased risk of death by 31% (95% confidence interval 28% to 33%, multivariate adjusted). Higher body mass index was associated with lower mortality in a J-shaped pattern with highest risk in underweight and normal weight veterans. Improved survival in obesity has been referred to as the obesity paradox in heart failure and other diseases. These data show that lower weight and diabetes are strong risk factors for mortality in PH. Our results underscore the importance of systemic conditions on outcome in PH.

Development of a small animal model for deer tick virus pathogenesis mimicking human clinical outcome.

Powassan virus (POWV) is a tick-borne flavivirus that encompasses two genetic lineages, POWV (Lineage I) and deer tick virus (DTV, Lineage II). In recent years, the incidence of reported POWV disease cases has increased, coupled with an expanded geographic range of the DTV tick vector, Ixodes scapularis. POWV and DTV are serologically indistinguishable, and it is not known whether clinical manifestations, pathology, or disease outcome differ between the two viruses. Six-week-old male and female BALB/c mice were footpad-inoculated with DTV doses ranging from 101 to 105 FFU. Dose-independent mortality, morbidity, and organ viral loads were observed for mice inoculated with sequentially increasing doses of DTV. By study completion, all surviving mice had cleared their viremias but detectable levels of negative-sense DTV RNA were present in the brain, indicating viral persistence of infectious DTV in the central nervous system. For mice that succumbed to disease, neuropathology revealed meningoencephalitis characterized by microscopic lesions with widespread distribution of viral RNA in the brain. These findings, coupled with the rapid onset of neurological signs of disease and high viral titers in nervous tissue, highlight the neurotropism of DTV in this mouse model. Additionally, disease outcome for DTV-infected mice was not affected by sex, as males and females were equally susceptible to disease. This is the first study to comprehensively characterize the clinical disease outcome in a small animal model across a spectrum of POWV/DTV infection doses. Here, we developed a small animal model for DTV pathogenesis that mimics the manifestations of POWV disease in humans. Since it is currently not known whether DTV and POWV differ in their capacity to cause human disease, the animal model detailed in our study could be utilized in future comparative pathogenesis studies, or as a platform for testing the efficacy of vaccines, and anti-virals.

Tick-Borne Encephalitis Virus Infection Alters the Sialome of Ixodes ricinus Ticks During the Earliest Stages of Feeding.

Ticks are hematophagous arthropods that transmit a number of pathogens while feeding. Among these is tick-borne encephalitis virus (TBEV), a flavivirus transmitted by Ixodes ricinus ticks in the temperate zone of Europe. The infection results in febrile illness progressing to encephalitis and meningitis with a possibility of fatality or long-term neurological sequelae. The composition of tick saliva plays an essential role in the initial virus transmission during tick feeding. Ticks secrete a diverse range of salivary proteins to modulate the host response, such as lipocalins to control the itch and inflammatory response, and both proteases and protease inhibitors to prevent blood coagulation. Here, the effect of viral infection of adult females of Ixodes ricinus was studied with the goal of determining how the virus alters the tick sialome to modulate host tissue response at the site of infection. Uninfected ticks or those infected with TBEV were fed on mice and removed and dissected one- and 3-h post-attachment. RNA from the salivary glands of these ticks, as well as from unfed ticks, was extracted and subjected to next-generation sequencing to determine the expression of key secreted proteins at each timepoint. Genes showing statistically significant up- or down-regulation between infected and control ticks were selected and compared to published literature to ascertain their function. From this, the effect of tick viral infection on the modulation of the tick-host interface was determined. Infected ticks were found to differentially express a number of uncategorized genes, proteases, Kunitz-type serine protease inhibitors, cytotoxins, and lipocalins at different timepoints. These virus-induced changes to the tick sialome may play a significant role in facilitating virus transmission during the early stages of tick feeding.

Detection of Rickettsiae, Borreliae, and Ehrlichiae in Ticks Collected from Walker County, Texas, 2017-2018.

Cases of tick-borne diseases, including spotted fever rickettsioses, borreliosis, babesiosis, anaplasmosis and ehrlichiosis, in the United States and territories have more than doubled from 2004 to 2016 and account for 77% of all vector-borne disease reports. In an effort to inform control efforts, the presence of tick-borne pathogens and their vectors was assessed in a recreational park in Walker County, Texas. Here we report data from questing ticks collected on three dates from June 2017 to June 2018. The majority of ticks collected were Amblyomma americanum (96.69%) followed by three additional tick species: Dermacentor variabilis (2.59%), Ixodes scapularis (0.52%), and A. maculatum (0.21%). Ticks were pooled and tested for molecular evidence of bacterial and viral pathogens, respectively. All of the 68 pools of A. americanum had molecular evidence of the spotted fever group rickettsia, Rickettsia amblyommatis. Additionally, six (8.82%) of the A. americanum pools contained sequences matching Ehrlichia chaffeensis, the pathogen responsible for human monocytotropic ehrlichiosis, and 11 (16.18%) for E. ewingii. Three of the A. americanum pools demonstrated evidence of Borrelia lonestari. The presence of etiologic agents of known human disease in this study merits the continued surveillance efforts of ticks and their pathogens in areas where they could pose risks to public health.

Functional CRISPR and shRNA Screens Identify Involvement of Mitochondrial Electron Transport in the Activation of Evofosfamide.

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ 0 (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.

Evofosfamide for the treatment of human papillomavirus-negative head and neck squamous cell carcinoma.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line-derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.

Metabolic and Physiologic Imaging Biomarkers of the Tumor Microenvironment Predict Treatment Outcome with Radiation or a Hypoxia-Activated Prodrug in Mice.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302.Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783-92. ©2018 AACR.